Method for producing the sesquiterpene viridiflorol with a fungal enzyme

ABSTRACT

Constructs, host cells, fungi, seeds, plants, and methods are described herein can include a Serendipita indica terpenoid synthase (SiTPS). Such constructs host cells, fungi, seeds, plants, and methods are useful, for example, for making viridiflorol. As described herein, the basidionycete Serendipita indica, a non-specific-host root endophyte fungus, possesses a functional terpenoid synthase gene (SiTPS). Heterologous expression of SiTPS in host cells showed that the produced protein efficiently utilizes the fifteen-carbon precursor farnesylpyrophosphate (FTP) to synthesize the sesquiterpene alcohol viridiflorol, shown below.

This application claims benefit of priority to the filing date of U.S.Provisional Application Ser. No. 62/899,391, filed Sep. 12, 2019, thecontents of which are specifically incorporated herein by reference intheir entirety.

BACKGROUND

Fungi may be source of bioactive and structurally diverse terpenoids.However, little is known about the genes responsible for theconstruction of these unique fungal terpenoid structures.

SUMMARY

As described herein, the basidiomycete Serendipita indica, anon-specific-host root endophyte fungus, possesses a functionalterpenoid synthase gene (SiTPS).

Heterologous expression of SiTPS in host cells showed that the producedprotein efficiently utilizes the fifteen-carbon precursorfarnesylpyrophosphate (FPP) to synthesize the sesquiterpene alcoholviridiflorol, shown below.

Viridiflorol has a highly characteristic scent and has utility in theperfume industry. It is also useful as bioactive molecule, withantibacterial, anti-inflammatory and antioxidant activities. Forexample, viridiflorol has antimicrobial activity against tuberculosis orother clinically relevant strains of multi-drug resistant (MDR)bacteria. Viridiflorol is found in essential oils of a variety of plantsincluding Melaleuca quinquenervia (broad-leaved paperbark), Melaleucaalternifolia (tea tree), and Allophylus edulis. This is the first reportof a SiTPS gene encoding a viridiflorol synthase.

Described herein are expression cassettes that include a heterologouspromoter operably linked to a nucleic acid segment encoding aSerendipita indica terpenoid synthase (SiTPS), where the Serendipitaindica terpenoid synthase can synthesize viridiflorol.

Described herein are host cells that have one or more are expressioncassettes that include a heterologous promoter operably linked to anucleic acid segment encoding a Serendipita indica terpenoid synthase(SiTPS), where the Serendipita indica terpenoid synthase can synthesizeviridiflorol.

Also described herein are fungi, spores, plants and seeds that have oneor more expression cassettes, each expression cassette having aheterologous promoter operably linked to a nucleic acid segment encodinga Serendipita indica terpenoid synthase (SiTPS), where the Serendipitaindica terpenoid synthase can synthesize viridiflorol.

Also described herein are methods for synthesizing viridiflorol thatinclude incubating one or more host cells that have one or moreexpression cassettes, each expression cassette having a heterologouspromoter operably linked to a nucleic acid segment encoding aSerendipita indica terpenoid synthase (SiTPS), where the Serendipitaindica terpenoid synthase can synthesize viridiflorol.

Also described herein are methods for synthesizing viridiflorol thatinclude cultivating one or more seeds that have one or more expressioncassettes, each expression cassette having a heterologous promoteroperably linked to a nucleic acid segment encoding a Serendipita indicaterpenoid synthase (SiTPS), where the Serendipita indica terpenoidsynthase can synthesize viridiflorol.

DESCRIPTION OF THE FIGURES

FIG. 1A-1B illustrate several biosynthetic pathways of sesquiterpenoids.FIG. 1A-1 to 1A-4 show biosynthetic pathways of several sesquiterpenoidsfound in Basidiomycota fungi. FIG. 1A-1 illustrates biosynthesis ofE,E-FPP and (3R)-NPP. Sesquiterpenoid scaffolds can be distinguished bythe first cyclization step of E,E-FPP or (3R)-NPP. Four distinctstructural groups can be made. FIG. 1A-2 illustrates biosynthesis ofterpenoids derived from 1,10 closure of E,E-FPP (STS Clade 1), and 1,11closure of E,E-FPP shown in blue (STS Clade III). FIG. 1A-3 illustratesbiosynthesis of terpenoids derived from 1,6 or 17 closure of (3R)-NPP(STS Clade IV). FIG. 1A-4 illustrates biosynthesis of terpenoids derivedfrom 1,10 closure of (3R)-NPP shown in blue (STS Clade II) (see also,Wawrzyn et al., 2012b). FIG. 1B-1 to 1B-4 illustrate a biosyntheticpathway leading to the production of viridiflorol and ledol. FIG. 1B-1shows that viridiflorol biosynthesis can start with 1,10-closure ofeither NPP (route a-Clade 11) or FPP (route b-Clade I) to form agermacradienyl cation (Z,E or EE, respectively). FIG. 1B-2 illustrates a1,3 H-shift (C1 to C11) of the germacradienyl cation. FIG. 1B-3illustrates two cyclisation events (1,11 and 2,6) of the H-shiftedgermacradienyl cation in the biosynthetic pathway leading to theproduction of viridiflorol and ledol. FIG. 1B-4 illustrates addition ofa molecule of water to the terpenoid generated as shown in FIG. 1B-3 ,thereby resulting in formation of viridiflorol and ledol (see also, Hong& Tantillo, 2011).

FIG. 2 illustrates Serendipita indica terpenoid synthase (SiTPS)phylogenetic relationships. A Maximum-likelihood tree was constructed(bootstrap of 100) using the proteins from NCBI with the highestsimilarity to SiTPS amino acid sequence. The scale bar indicates agenetic distance of 0.50 and the bootstrap values are shown below thebranches. NCBI accession numbers: CCA72799.1 Serendipita indica DSM11827, KIM31075.1 Serendipita vermifera MAFF 305830, K1032332.1Tulasnella calospora MUT 4182, AUW30846.1 Cladonia uncialis subspuncialis, KDQ10835.1 Botrvobasidium botryosum, CEL57650.1 Rhizoctoniasolani AG-1 IB, CUA75792.1 Rhizoetonia solani, XP_007869182.1Gloeophyllum trabeum ATCC 11539, OBZ72577.1 Grifola frondosa,WP_073739522.1 Streptomyces sp, WP_030800135.1 Streptomycesalbovinaceus, SCG69967.1 Micromonospora zamorensis, SFC79096.1Verrucosispora sediminis, SCG62678.1 Micromonospora coxensis, SCL17011.1Micromonospora rhizosphaerae, SBV28600.1 Micromonospora krabiensis,WP_007454512.1 Micromonospora lupine, SCL47036.1 Micromonosporayangpuensis, WP_013730983.1 Verrucosispora maris.

FIG. 3A-B illustrates in vivo and in vitro characterization of assay forSiTPS enzyme activity, as detected by gas chromatography-massspectroscopy (GC-MS) analysis of hexane extracts of the assay reactions.The in vitro assay included incubation of the purified SiTPS with thesubstrate FPP, while the in vivo assay included use of E. coli strainstransformed with the plasmids pIRS, pFF and pET_SiTPS. An E. coli straincarrying only the pIRS and pFF plasmids was used as control. Analyticalstandards of viridiflorol and ledol are also shown.

FIG. 4 graphically illustrates the relative expression of SITPS when S.indica is grown for 14 days on CM agar plates (in CM, lighter bar)compared to expression when S. indica fungi is colonizing tomato roots(11 days post inoculation, dpi) (in planta, darker bar). Relativeexpression in both conditions was normalized using SiGAPDH (GenBank:FJ810523.1) expression levels and calculated with the 2^(−ΔCt) method(Livak & Schmittgen, 2001; Schmittgen & Livak, 2008). Error barsrepresent the standard deviation (n=4). Asterisk (*) representssignificant difference between the two conditions (t-test in Rstudio,*P-value≤0.05).

FIG. 5 graphically illustrates the colonisation ability of S. indicawild type (wt, left bars), S. indica expressing an empty vector (evV,middle bars), and S. indica over-expressing the SiTPS enzyme (ovII,right bars) at 2 days post inoculation (dpi). To measure the ability ofto colonise plant roots the colonisation ratio, the ratio of fungal toplant DNA (DNAf/DNAp), was used. No statistical difference was observedbetween the different treatments (one-way ANOVA).

FIG. 6 is a schematic diagram illustrating the structure of the plasmidK167_PromFCGBI_SiTPS_His used for creating the SiTPS-overexpressingmutants. The vector backbone is comprised of a hygromycin markercassette and an ampicillin-resistance gene. The SiTPS sequence is HA-and His-tagged. Gene expression is under the control of the promoterFCGB1 of S. indica.

FIG. 7 graphically illustrates the relative expression of SiTPS in thefollowing S. indica strains wild type (wt, left bar); empty vectorstrain-transformant V (evV, middle bar); and SiTPS-overexpressingstrain-transformant 11 (ovII, right bar) after growth for 14 days on CMagar plates. Relative expression was normalized using SiGAPDH (GenBank:FJ810523.1) expression levels and calculated with the 2^(−ΔCt) method(Livak & Schmittgen, 2001; Schmittgen & Livak, 2008). Error barsrepresent the standard deviation (n=4). Different letters indicatestatistical differences (t-test in Rstudio, P-value≤0.05).

DETAILED DESCRIPTION

Constructs, host cells, seeds, plants, and methods are described hereinthat include a Serendipita indica terpenoid synthase (SiTPS). Suchconstructs, host cells, seeds, plants, and methods are useful for makingterpenoids such as viridiflorol.

Serendipita indica Terpenoid Synthase (SiTPS)

Serendipita (also called Piriformospora) indica is a fungal endophyticsymbiont with the capabilities to enhance plant growth and conferresistance to different stresses. However, the application of thisfungus in the field has led to inconsistent results. An enzyme fromSerendipita indica (SiTPS) is described herein that is useful for makingterpenoids such as viridiflorol. Volatile terpenoids can mediatecommunication between plants and microbes and plant terpenoids areinvolved in the development of ectomycorrhizal associations with severalplant species during the pre-symbiotic phase. Despite a couple ofattempts (Crutcher et al., 2013), there has previously been no directevidence that fungal terpenoids could play a role in facilitatingestablishment of plant-endophytic interactions.

The enzyme from Serendipita indica (SiTPS) useful for making terpenoidsis referred to as the SiTPS enzyme. This enzyme can have the followingamino acid sequence (SEQ ID NO:1).

  1 MPSVSPATIR LPDILGAMDR FELRTHPDER EVTRASNEWF  41NSYNMMPPAL FEKFVKCDFG LMTGMSYPDT DATRLRITCD  81YMSILFAYDD LMDLPSSDLM HDKIASDKAA KIMMGVLTHP 121HKFRPYAGLP VATAFHDFWT RFCATSTPKM QKRFTDTTYE 161YVMAVKNQCG NRQSSRCPTI EEYVALRRDT SAIKVTYACI 201EYCLNIDVPD EAFYHPSVAA LQEAGNNILS WANDVYSFDN 241EQSSGDCHNL VAIVAINKNI TVQAAMEYVM GMIDSAIERF 281FEECANVPSF GPEVDPLVQA YIKGVELYLS GSVFWHLESE 321RYFGARVQHV KDTLMVELRP LDEGAKPAFD LMYKLPSNLT 361PEVLSAAAVS AAPAAPAPVA SPAPQPEILS PTPISPINVN 401FPLGNVACPP PSYETQRVLA KMVAATVEEK QRLAYSQPAE 441QYYSPAPQYY PSQPVEKFQQ TNVLETAFKG SNSELTNILV 481IASVLMAGSP MALVPFVPLL ALLLLPNETP VAPVAFEHHH 521 HHHA nucleic acid sequence that encodes the SEQ ID NO:1 SiTPS is shownbelow as SEQ ID NO:2.

1 ATGCCATCTG TCTCTCCTGC CACCATCCGC CTCCCTGATA 41TCCTCGGTGC TATGGACCGC TTTGAGCTCC GCACTCACCC 81CGATGAGCGC GAAGTCACCC GTGCCTCGAA CGAGTGGTTC 121AACAGCTACA ACATGATGCC CCCGGCACTC TTTGAAAAGT 161TTGTCAAGTG CGATTTCGGC CTCATGACCG GCATGTCGTA 201CCCAGATACC GATGCTACCC GCCTCCGTAT CACTTGCGAC 241TACATGTCGA TCCTCTTCGC CTATGACGAC CTCATGGACC 281TCCCCTCGTC CGACCTTATG CATGACAAGA TTGCCTCGGA 321CAAGGCTGCC AAGATCATGA TGGGTGTCCT CACCCACCCC 361CACAAGTTCC GCCCATATGC TGGCCTCCCA GTCGCCACTG 401CTTTCCATGA CTTCTGGACC CGCTTCTGCG CTACTTCGAC 441CCCAAAGATG CAAAAGCGCT TCACTGACAC CACCTACGAG 481TATGTCATGG CCGTCAAGAA CCAGTGCGGC AACCGCCAGA 521GCTCTCGCTG CCCAACCATC GAGGAGTACG TCGCTCTCCG 561CCGCGACACC TCGGCCATCA AGGTCACCTA TGCTTGCATC 601GAGTACTGCC TCAACATCGA CGTCCCAGAC GAGGCCTTCT 641ACCACCCCTC CGTGGCTGCT CTCCAGGAGG CTGGCAATAA 681TATCCTCTCG TGGGCCAACG ATGTTTACTC GTTTGACAAC 721GAGCAATCCT CGGGTGACTG CCACAACCTC GTTGCCATTG 761TTGCCATCAA CAAGAACATT ACTGTTCAGG CTGCAATGGA 801GTACGTCATG GGCATGATCG ACTCTGCTAT CGAACGCTTC 841TTCGAGGAGT GCGCCAACGT CCCTTCGTTC GGCCCCGAAG 881TCGACCCTCT CGTCCAGGCC TACATCAAGG GTGTCGAGCT 921CTACCTTAGC GGCTCCGTCT TCTGGCACCT CGAATCCGAG 961CGCTACTTTG GTGCTCGCGT CCAGCACGTC AAGGATACCT 1001TGATGGTTGA GCTCCGCCCA CTCGACGAGG GTGCGAAGCC 1041GGCCTTCGAC CTCATGTACA AGCTCCCATC CAACTTGACG 1081CCCGAGGTCC TCAGTGCCGC TGCTGTCTCG GCTGCCCCAG 1121CTGCGCCAGC TCCTGTCGCT TCTCCGGCTC CTCAGCCAGA 1161GATCCTCTCG CCGACGCCAA TCTCGCCCAT CAACGTCAAC 1201TTCCCTCTCG GCAACGTCGC CTGCCCGCCT CCTTCGTACG 1241AGACCCAGCG CGTTCTCGCC AAGATGGTGG CCGCGACCGT 1281CGAGGAGAAG CAGCGCCTTG CTTAGAGCCA GCCAGCTGAG 1321CAGTACTACT CGCCCGCTCC CCAGTACTAC CCAAGCCAGC 1361CGGTTGAAAA GTTCCAGCAG ACCAACGTGC TCGAGACCGC 1401CTTCAAGGGA TCCAACTCGG AATTGACCAA CATTCTCGTT 1441ATTGCCTCCG TCCTCATGGC CGGATCACCC ATGGCGCTTG 1481TCCCCTTTGT CCCTCTTCTC GCCCTCCTAC TCCTCCCCAA 1521CGAGACCCCA GTGGCTCCCG TTGCGTTCGA GCACCACCAC 1561 CACCACCACThe SiTPS enzyme can have one or more amino acids missing from the SEQID NO:1 sequence. For example, the SiTPS enzyme can have the C-terminalhistidine tag (HHHHHH at positions 518-523) missing. Such a SiTPS enzymecan have the following sequence (SEQ ID NO: 3).

  1 MPSVSPATIR LPDILGAMDR FELRTHPDER EVTRASNEWF  41NSYNMMPPAL FEKFVKCDFG LMTGMSYPDT DATRLRITCD  81YMSILFAYDD LMDLPSSDLM HDKIASDKAA KIMMGVLTHP 121HKFRPYAGLP VATAFHDFWT RFCATSTPKM QKRFTDTTYE 161YVMAVKNQCG NRQSSRCPTI EEYVALRRDT SAIKVTYACI 201EYCLNIDVPD EAFYHPSVAA LQEAGNNILS WANDVYSFDN 241EQSSGDCHNL VAIVAINKNI TVQAAMEYVM GMIDSAIERF 281FEECANVPSF GPEVDPLVQA YIKGVELYLS GSVEWHDESE 321RYFGARVQHV KDTLMVELRP LDEGAKPAFD LMYKLPSNLT 361PEVLSAAAVS AAPAAPAPVA SPAPQPEILS PTPISPINVN 401FPLGNVACPP PSYETQRVLA KMVAATVEEK QRLAYSQPAE 441QYYSPAPQYY PSQPVEKFQQ TNVLETAFKG SNSELTNILV 481IASVLMAGSP MALVPFVPLL ALLLLPNETP VAPVAFE

In another example, the SiTPS enzyme can also have the following aminoacid sequence (SEQ ID NO:4), where there is one amino acid missing atabout position 218.

  1 MPSVSPATIR LPDILGAMDR FELRTHPDER EVTRASNEWF  41NSYNMMPPAL FEKFVKCDFG LMTGMSYPDT DATRLRITCD  81YMSILFAYDD LMDLPSSDLM HDKIASDKAA KIMMGVLTHP 121HKFRPYAGLP VATAFHDFWT RFCATSTPKM QKRFTDTTYE 161YVMAVKNQCG NRQSSRCPTI EEYVALRRDT SAIKVTYACI 201EYCLNIDVPD EAFYHPSVAA LQEAGNILS  WANDVYSFDN 241EQSSGDCHNL VAIVAINKNI TVQAAMEYVM GMIDSAIERF 281FEECANVPSF GPEVDPLVQA YIKGVELYLS GSVFWHLESE 321RYFGARVQHV KDTLMVELRP LDEGAKPAFD LMYKLPSNLT 361PEVLSAAAVS AAPAAPAPVA SPAPQPEILS PTPISPINVN 401FPLGNVACPP PSYETQRVLA KMVAATVEEK QRLAYSQPAE 441QYYSPAPQYY PSQPVEKFQQ TNVLETAFKG SNSELTNILV 481IASVLMAGSP MALVPFVPLL ALLLLPNETP VAPVAFEHHH 521 HHHThe SiTPS enzyme with SEQ ID NO:4 can also not have the C-terminalhistidine tag (HHHHHH, SEQ ID NO:6) at positions 518-523.

Enzymes described herein can have one or more deletions, insertions,replacements, or substitutions in a part of the enzyme. The enzyme(s)described herein can have, for example, at least 60%, or at least 70%,or at least 80%, or at least 90%, or at least 93%, or at least 95%, orat least 96%, or at least 97%, or at least 98%, or at least 99% sequenceidentity to a sequence described herein.

Nucleic acids encoding one or more enzyme(s) can have one or morenucleotide deletions, insertions, replacements, or substitutions. Forexample, the nucleic acids encoding one or more enzyme(s) can, forexample, have less than 95%, or less than 94.8%, or less than 94.5%, orless than 94%, or less than 93.8%, or less than 94.50% nucleic acidsequence identity to a corresponding parental or wild-type sequence. Insome cases, the nucleic acids encoding one or more enzyme(s) can have,for example, at least 50%, or at least 55%, or at least 60%, or at least65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%,or at 90% sequence identity to a corresponding parental or wild-typesequence.

In some cases, enzymes can have conservative changes such as one or moredeletions, insertions, replacements, or substitutions that have nosignificant effect on the activities of the enzymes. Examples ofconservative substitutions are provided below in Table 1A.

TABLE 1A Conservative Substitutions Type of Amino Acid SubstitutableAmino Acids Hydrophilic Ala, Pro, Gly, Glu, Asp, Gln, Asn, Ser, ThrSulfhydryl Cys Aliphatic Val, Ile, Leu, Met Basic Lys, Arg, His AromaticPhe, Tyr, Trp

Synthetic Pathways

Biosynthetic pathways of several sesquiterpenoids found in Basidiomycotafungi are shown below. For example, the mevalonate (MVA) pathway isshown below (see also FIG. 1A), which provides isopentenyl diphosphate(IPP) and dimethylallyl diphosphate building (DMAPP) building blocks forbiosynthesis of isoprenoids.

For example, the isopentenyl diphosphate (IPP) and dimethylallyldiphosphate building (DMAPP) are building blocks for E,E-FPP and(3R)-NPP, which are building blocks for synthesis of viridiflorol.

The E,E-FPP or (3R)-NPP precursors are converted to viridiflorol asshown below.

Viridiflorol biosynthesis starts with a 1,10-closure of either NPP(route a-Clade 11) or FPP (route b-Clade I) to form a germacradienylcation (Z,E or E,E respectively). Two more cyclisation events (1,11 and2,6) and the addition of a molecule of water result in the formation ofviridiflorol.

Expression of Enzymes

Also described herein are expression systems that include at least oneexpression cassette (e.g., expression vectors or transgenes) that encodeone or more of the enzyme(s) described herein. The expression systemscan also include one or more expression cassettes encoding an enzymethat can synthesize terpene building blocks. For example, the expressionsystems can include one or more expression cassettes encoding terpenesynthases that facilitate production of terpene precursors or buildingblocks such as those involved in the synthesis of isopentenyldiphosphate (IPP) or dimethylallyl diphosphate (DMAPP).

Cells containing such expression systems are further described herein.The cells containing such expression systems can be used to manufacturethe enzymes (e.g., for in vitro use) and/or one or more terpenes,diterpenes, sesquiterpenes, or terpenoids produced by the enzymes.Methods of using the enzymes or cells containing expression cassettesencoding such enzymes to make products such as terpenes, diterpenes,terpenoids, and combinations thereof are also described herein.

Nucleic acids encoding the enzymes can have sequence modifications. Forexample, nucleic acid sequences described herein can be modified toexpress enzymes that have modifications. Most amino acids can be encodedby more than one codon. When an amino acid is encoded by more than onecodon, the codons are referred to as degenerate codons. A listing ofdegenerate codons is provided in Table 1B below.

TABLE 1B Degenerate Amino Acid Codons Amino Acid Three Nucleotide CodonAla/A GCT, GCC, GCA, GCG Arg/R CGT, CGC, CGA, CGG, AGA, AGG Asn/NAAT, AAC Asp/D GAT, GAC Cys/C TGT, TGC Gln/Q CAA, CAG Glu/E GAA, GAGGly/G GGT, GGC, GGA, GGG His/H CAT, CAC Ile/I ATT, ATC, ATA Leu/LTTA, TTG, CTT, CTC, CTA, CTG Lys/K AAA, AAG Met/M ATG Phe/F TTT, TTCPro/P CCT, CCC, CCA, CCG Ser/S TCT, TCC, TCA, TCG, AGT, AGC Thr/TACT, ACC, ACA, ACG Trp/W TGG Tyr/Y TAT, TAC Val/V GTT, GTC, GTA, GTGSTART ATG STOP TAG, TGA, TAA

Different organisms may translate different codons more or lessefficiently (e.g., because they have different ratios of tRNAs) thanother organisms. Hence, when some amino acids can be encoded by severalcodons, a nucleic acid segment can be designed to optimize theefficiency of expression of an enzyme by using codons that are preferredby an organism of interest. For example, the nucleotide coding regionsof the enzymes described herein can be codon optimized for expression invarious plant species. For example, many of the enzymes described hereinwere originally isolated from the mint family (Lamiaceae), however suchenzymes can be expressed in a variety of host cells, including forexample, bacterial, fungal, and plant host cells.

An optimized nucleic acid can have less than 98%, less than 97%, lessthan 95%, or less than 94%, or less than 93%, or less than 92%, or lessthan 91%, or less than 90%, or less than 89%, or less than 88%, or lessthan 85%, or less than 83%, or less than 80%, or less than 75% nucleicacid sequence identity to a corresponding non-optimized (e.g., anon-optimized parental or wild type enzyme nucleic acid) sequence.

The enzymes described herein can be expressed from an expressioncassette and/or an expression vector. Such an expression cassette caninclude a nucleic acid segment that encodes an enzyme operably linked toa promoter to drive expression of the enzyme. Convenient vectors, orexpression systems can be used to express such enzymes. In someinstances, the nucleic acid segment encoding an enzyme is operablylinked to a promoter and/or a transcription termination sequence. Thepromoter and/or the termination sequence can be heterologous to thenucleic acid segment that encodes an enzyme. Expression cassettes canhave a promoter operably linked to a heterologous open reading frameencoding an enzyme. The invention therefore provides expressioncassettes or vectors useful for expressing one or more enzyme(s).

Constructs, e.g., expression cassettes, and vectors comprising theisolated nucleic acid molecule, e.g., with optimized nucleic acidsequence, as well as kits comprising the isolated nucleic acid molecule,construct or vector are also provided.

The nucleic acids described herein can also be modified to improve oralter the functional properties of the encoded enzymes. Deletions,insertions, or substitutions can be generated by a variety of methodssuch as, but not limited to, random mutagenesis and/or site-specificrecombination-mediated methods. The mutations can range in size from oneor two nucleotides to hundreds of nucleotides (or any value therebetween). Deletions, insertions, and/or substitutions are created at adesired location in a nucleic acid encoding the enzyme(s).

Nucleic acids encoding one or more enzyme(s) can have one or morenucleotide deletions, insertions, replacements, or substitutions. Forexample, the nucleic acids encoding one or more enzyme(s) can, forexample, have less than 95%, or less than 94.8%, or less than 94.5%, orless than 94%, or less than 93.8%, or less than 94.50% nucleic acidsequence identity to a corresponding parental or wild-type sequence. Insome cases, the nucleic acids encoding one or more enzyme(s) can have,for example, at least 50%, or at least 55%, or at least 60%, or at least65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%,or at 90% sequence identity to a corresponding parental or wild-typesequence. An example of a parental or wild type nucleic acid sequencesfor unmodified enzyme(s) with amino acid sequence SEQ ID NOs:1 and 3, isnucleic acid sequence SEQ ID NO:2.

Any of these nuclei acid or amino acid sequences can, for example,encode or have enzyme sequences with less than 99%, less than 98%, lessthan 97%, less than 96%, less than 95%, less than 94.8%, less than94.5%, less than 94%, less than 93.8%, less than 93.5%, less than 93%,less than 92%, less than 91%, or less than 90% sequence identity to acorresponding parental or wild-type sequence.

Also provided are nucleic acid molecules (polynucleotide molecules) thatcan include a nucleic acid segment encoding an enzyme with a sequencethat is optimized for expression in at least one selected host organismor host cell. Optimized sequences include sequences which are codonoptimized, i.e., codons which are employed more frequently in oneorganism relative to another organism. In some cases, the balance ofcodon usage is such that the most frequently used codon is not used toexhaustion. Other modifications can include addition or modification ofKozak sequences and/or introns, and/or to remove undesirable sequences,for instance, potential transcription factor binding sites.

An enzyme useful for synthesis of terpenes, diterpenes, and terpenoidsmay be expressed on the surface of, or within, a prokaryotic oreukaryotic cell. In some cases, expressed enzyme(s) can be secreted bythat cell.

Techniques of molecular biology, microbiology, and recombinant DNAtechnology which are within the skill of the art can be employed to makeand use the enzymes, expression systems, and terpene products describedherein. Such techniques available in the literature. See, e.g.,Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual,Second Edition (1989); DNA Cloning, Vols. I and II (D. N. Glover ed.1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984): Animal CellCulture (R. K. Freshney ed. 1986); Immobilized Cells and Enzymes (IRLpress, 1986); Perbal, B., A Practical Guide to Molecular Cloning (1984);the series Methods In Enzymology (S. Colowick and N. Kaplan eds.,Academic Press, Inc.); Current Protocols In Molecular Biology (JohnWiley & Sons, Inc), Current Protocols In Protein Science (John Wiley &Sons, Inc), Current Protocols In Microbiology (John Wiley & Sons, Inc),Current Protocols In Nucleic Acid Chemistry (John Wiley & Sons, Inc),and Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C.C. Blackwell eds., 1986, Blackwell Scientific Publications).

Prokaryotic cells can be used as host cells to express the enzymes andproduce terpenes.

Modified plants and fungi that contain nucleic acids encoding enzymeswithin their somatic and/or germ cells are described herein. Suchgenetic modification can be accomplished by available procedures. Forexample, one of skill in the art can prepare an expression cassette orexpression vector that can express one or more encoded enzymes. Plantand fungal cells can be transformed by the expression cassette orexpression vector. Fungi can be generated from the fungal cells. Wholeplants (and their seeds) can be generated from the plant cells that weresuccessfully transformed with the enzyme nucleic acids. Some proceduresfor making such genetically modified plants and their seeds aredescribed below.

Promoters: The nucleic acids encoding enzymes can be operably linked toa promoter, which provides for expression of mRNA from the nucleic acidsencoding the enzymes. The promoter is typically a promoter functional inplants and can be a promoter functional during plant growth anddevelopment. A nucleic acid segment encoding an enzyme is operablylinked to the promoter when it is located downstream from the promoter.The combination of a coding region for an enzyme operably linked to apromoter forms an expression cassette, which can optionally includeother elements as well.

Promoter regions are typically found in the flanking DNA upstream fromthe coding sequence in both the prokaryotic and eukaryotic cells. Apromoter sequence provides for regulation of transcription of thedownstream gene sequence and typically includes from about 50 to about2,000 nucleotide base pairs. Promoter sequences also contain regulatorysequences such as enhancer sequences that can influence the level ofgene expression. Some isolated promoter sequences can provide for geneexpression of heterologous DNAs, that is a DNA different from the nativeor homologous DNA.

Promoter sequences are also known to be strong or weak, or inducible. Astrong promoter provides for a high level of gene expression, whereas aweak promoter provides for a very low level of gene expression. Aninducible promoter is a promoter that provides for the turning geneexpression on and off in response to an exogenously added agent, or toan environmental or developmental stimulus. For example, a bacterialpromoter such as the P_(tac) promoter can be induced to varying levelsof gene expression depending on the level ofisopropyl-beta-D-thiogalactoside added to the transformed cells.Promoters can also provide for tissue specific or developmentalregulation. An isolated promoter sequence that is a strong promoter forheterologous DNAs is advantageous because it provides for a sufficientlevel of gene expression for easy detection and selection of transformedcells and provides for a high level of gene expression when desired.

Expression cassettes generally include, but are not limited to, examplesof plant promoters such as the CaMV 35S promoter (Odell et al., Nature.313:810-812 (1985)), or others such as CaMV 19S (Lawton et al., PlantMolecular Biology. 9:315-324 (1987)), nos (Ebert et al., Proc. NatL.Acad. Sci. USA. 84:5745-5749 (1987)), Adh1 (Walker et al., Proc. Natl.Acad. Sci. USA. 84:6624-6628 (1987)), sucrose synthase (Yang et al.,Proc. Natl. Acad. Sci. USA. 87:4144-4148 (1990)), α-tubulin, ubiquitin,actin (Wang et al., Mol. Cell. Biol. 12:3399 (1992)), cab (Sullivan etal., Mol. Gen. Genet. 215:431 (1989)), PEPCase (Hudspeth et al., PlantMolecular Biology. 12:579-589 (1989)) or those associated with the Rgene complex (Chandler et al., The Plant Cell. 1:1175-1183 (1989)).Further suitable promoters include a CYP71D16 trichome-specific promoterand the CBTS (cembratrienol synthase) promotor, cauliflower mosaic viruspromoter, the Z10 promoter from a gene encoding a 10 kD zein protein, aZ27 promoter from a gene encoding a 27 kD zein protein, the plastidrRNA-operon (rrn) promoter, inducible promoters, such as the lightinducible promoter derived from the pea rbcS gene (Coruzzi et al., EMBOJ. 3:1671 (1971)), RUBISCO-SSU light inducible promoter (SSU) fromtobacco and the actin promoter from rice (McElroy et al., The PlantCell. 2:163-171 (1990)). Other promoters that are useful can also beemployed.

Alternatively, novel tissue specific promoter sequences may be employed.cDNA clones from a particular tissue can be isolated and those cloneswhich are expressed specifically in that tissue can be identified, forexample, using Northern blotting. Preferably, the gene isolated is notpresent in a high copy number but is relatively abundant in specifictissues. The promoter and control elements of corresponding genomicclones can then be localized using techniques well known to those ofskill in the art.

A nucleic acid encoding an enzyme can be combined with the promoter bystandard methods to yield an expression cassette, for example, asdescribed in Sambrook et al. (MOLECULAR CLONING: A LABORATORY MANUAL.Second Edition (Cold Spring Harbor, N.Y.: Cold Spring Harbor Press(1989); MOLECULAR CLONING: A LABORATORY MANUAL. Third Edition (ColdSpring Harbor, N.Y.: Cold Spring Harbor Press (2000)). Briefly, aplasmid containing a promoter such as the 35S CaMV promoter or theCYP71D16 trichome-specific promoter can be constructed as described inJefferson (Plant Molecular Biology Reporter 5:387-405 (1987)) orobtained from Clontech Lab in Palo Alto, Calif. (e.g., pBI121 orpBI221). Typically, these plasmids are constructed to have multiplecloning sites having specificity for different restriction enzymesdownstream from the promoter.

The nucleic acid sequence encoding for the enzyme(s) can be subcloneddownstream from the promoter using restriction enzymes and positioned toensure that the DNA is inserted in proper orientation with respect tothe promoter so that the DNA can be expressed as sense RNA. Once thenucleic acid segment encoding the enzyme is operably linked to apromoter, the expression cassette so formed can be subcloned into aplasmid or other vector (e.g., an expression vector).

In some embodiments, a cDNA clone encoding an enzyme is isolated from amint species, for example, from leaf, trichome, or root tissue. In otherembodiments, cDNA clones from other species (that encode an enzyme) areisolated from selected plant tissues, or a nucleic acid encoding a wildtype, mutant or modified enzyme is prepared by available methods or asdescribed herein. For example, the nucleic acid encoding the enzyme canbe a nucleic acid with a coding region that hybridizes to SEQ ID NO:2,and that has enzyme activity. Using restriction endonucleases, theentire coding sequence for the enzyme is subcloned downstream of thepromoter in a 5′ to 3′ sense orientation.

Targeting Sequences: Additionally, expression cassettes can beconstructed and employed to target the nucleic acids encoding an enzymeto an intracellular compartment within plant cells or to direct anencoded protein to the extracellular environment. This can generally beachieved by joining a DNA sequence encoding a transit or signal peptidesequence to the coding sequence of the nucleic acid encoding the enzyme.The resultant transit, or signal, peptide can transport the protein to aparticular intracellular, or extracellular, destination and can then beco-translationally or post-translationally removed. Transit peptides actby facilitating the transport of proteins through intracellularmembranes, e.g., vacuole, vesicle, plastid and mitochondrial membranes,whereas signal peptides direct proteins through the extracellularmembrane. By facilitating transport of the protein into compartmentsinside or outside the cell, these sequences can increase theaccumulation of a particular gene product within a particular location.For example, see U.S. Pat. No. 5,258,300.

For example, in some cases it may be desirable to localize the enzymesto the plastidic compartment and/or within plant cell trichomes. Thebest compliment of transit peptides/secretion peptide/signal peptidescan be empirically ascertained. The choices can range from using thenative secretion signals akin to the enzyme candidates to betransgenically expressed, to transit peptides from proteins known to belocalized into plant organelles such as trichome plastids in general.For example, transit peptides can be selected from proteins that have arelative high titer in the trichomes. Examples include, but not limitedto, transit peptides form a terpenoid cyclase (e.g. cembratrieneolcyclase), the LTP1 protein, the Chlorophyll a-b binding protein 40,Phylloplanin, Glycine-rich Protein (GRP), Cytochrome P450 (CYP71D16);all from Nicotiana sp. alongside RUBISCO (Ribulose bisphosphatecarboxylase) small unit protein from both Arabidopsis and Nicotiana sp.

3′ Sequences: When the expression cassette is to be introduced into aplant cell, the expression cassette can also optionally include 3′untranslated plant regulatory DNA sequences that act as a signal toterminate transcription and allow for the polyadenylation of theresultant mRNA. The 3′ untranslated regulatory DNA sequence can includefrom about 300 to 1,000 nucleotide base pairs and can contain planttranscriptional and translational termination sequences. For example, 3′elements that can be used include those derived from the nopalinesynthase gene of Agrobacterium tumefaciens (Bevan et al., Nucleic AcidResearch. 11:369-385 (1983)), or the terminator sequences for the T7transcript from the octopine synthase gene of Agrobacterium tumefaciens,and/or the 3′ end of the protease inhibitor I or II genes from potato ortomato. Other 3′ elements known to those of skill in the art can also beemployed. These 3′ untranslated regulatory sequences can be obtained asdescribed in An (Methods in Enzymology. 153:292 (1987)). Many such 3′untranslated regulatory sequences are already present in plasmidsavailable from commercial sources such as Clontech, Palo Alto, Calif.The 3′ untranslated regulatory sequences can be operably linked to the3′ terminus of the nucleic acids encoding the enzyme.

Selectable and Screenable Marker Sequences: To improve identification oftransformants, a selectable or screenable marker gene can be employedwith the expressible nucleic acids encoding the enzyme(s). “Markergenes” are genes that impart a distinct phenotype to cells expressingthe marker gene and thus allow such transformed cells to bedistinguished from cells that do not have the marker. Such genes mayencode either a selectable or a screenable marker, depending on whetherthe marker confers a trait which one can ‘select’ for by chemical means,i.e., through the use of a selective agent (e.g., a herbicide,antibiotic, or the like), or whether it is simply a trait that one canidentify through observation or testing, i.e., by ‘screening’ (e.g., theR-locus trait). Of course, many examples of suitable marker genes areavailable can be employed in the practice of the invention.

Included within the terms ‘selectable or screenable marker genes’ arealso genes which encode a “secretable marker” whose secretion can bedetected as a means of identifying or selecting for transformed cells.Examples include markers which encode a secretable antigen that can beidentified by antibody interaction, or secretable enzymes that can bedetected by their catalytic activity. Secretable proteins fall into anumber of classes, including small, diffusible proteins detectable,e.g., by ELISA; and proteins that are inserted or trapped in the cellwall (e.g., proteins that include a leader sequence such as that foundin the expression unit of extensin or tobacco PR-S).

With regard to selectable secretable markers, the use of an expressionsystem that encodes a polypeptide that becomes sequestered in the cellwall, where the polypeptide includes a unique epitope may beadvantageous. Such a cell wall antigen can employ an epitope sequencethat would provide low background in plant tissue, a promoter-leadersequence that imparts efficient expression and targeting across theplasma membrane, and that can produce protein that is bound in the cellwall and yet is accessible to antibodies. A normally secreted cell wallprotein modified to include a unique epitope would satisfy suchrequirements.

Example of protein markers suitable for modification in this mannerinclude extensin or hydroxyproline rich glycoprotein (HPRG). Forexample, the maize HPRG (Stiefel et al., The Plant Cell. 2:785-793(1990)) is well characterized in terms of molecular biology, expression,and protein structure and therefore can readily be employed. However,any one of a variety of extensins and/or glycine-rich cell wall proteins(Keller et al., EMBO J. 8:1309-1314 (1989)) could be modified by theaddition of an antigenic site to create a screenable marker.

Selectable markers for use in connection with the present invention caninclude, but are not limited to, a neo gene (Potrykus et al., Mol. Gen.Genet. 199:183-188 (1985)) which codes for kanamycin resistance and canbe selected for using kanamycin, G418; a bar gene which codes forbialaphos resistance; a gene which encodes an altered EPSP synthaseprotein (Hinchee et al., Bio/Technology. 6:915-922 (1988)) thusconferring glyphosate resistance; a nitrilase gene such as bxn fromKlebsiella ozaenae which confers resistance to bromoxynil (Stalker etal., Science. 242:419-423 (1988)); a mutant acetolactate synthase gene(ALS) which confers resistance to imidazolinone, sulfonylurea or otherALS-inhibiting chemicals (European Patent Application 154,204 (1985)); amethotrexate-resistant DHFR gene (Thillet et al., J. Biol. Chem.263:12500-12508 (1988)); a dalapon dehalogenase gene that confersresistance to the herbicide dalapon; or a mutated anthranilate synthasegene that confers resistance to 5-methyl tryptophan. Where a mutant EPSPsynthase gene is employed, additional benefit may be realized throughthe incorporation of a suitable chloroplast transit peptide. CTP(European Patent Application 0 218 571 (1987)).

An illustrative embodiment of a selectable marker gene capable of beingused in systems to select transformants is the gene that encode theenzyme phosphinothricin acetyltransferase, such as the bar gene fromStreptomyces hygroscopicus or the pat gene from Streptomycesviridochromogenes (U.S. Pat. No. 5,550,318). The enzyme phosphinothricinacetyl transferase (PAT) inactivates the active ingredient in theherbicide bialaphos, phosphinothricin (PPT). PPT inhibits glutaminesynthetase. (Murakami et al., Mol. Gen. Genet. 205:42-50 (1986); Twellet al., Plant Physiol. 91:1270-1274 (1989)) causing rapid accumulationof ammonia and cell death. Screenable markers that may be employedinclude, but are not limited to, a β-glucuronidase or uidA gene (GUS)that encodes an enzyme for which various chromogenic substrates areknown; an R-locus gene, which encodes a product that regulates theproduction of anthocyanin pigments (red color) in plant tissues(Dellaporta et al., In: Chromosome Structure and Function: Impact of NewConcepts, 18^(th) Stadler Genetics Symposium, J. P. Gustafson and R.Appels, eds. (New York: Plenum Press) pp. 263-282 (1988)); a β-lactamasegene (Sutcliffe, Proc. Natl. Acad. Sci. USA. 75:3737-3741 (1978)), whichencodes an enzyme for which various chromogenic substrates are known(e.g., PADAC, a chromogenic cephalosporin); a xylE gene (Zukowsky etal., Proc. Natl. Acad. Sci. USA. 80:1101 (1983)) which encodes acatechol dioxygenase that can convert chromogenic catechols; anα-amylase gene (Ikuta et al., Bio/technology 8:241-242 (1990)); atyrosinase gene (Katz et al., J. Gen. Microbiol. 129:2703-2714 (1983))which encodes an enzyme capable of oxidizing tyrosine to DOPA anddopaquinone which in turn condenses to form the easily detectablecompound melanin; a β-galactosidase gene, which encodes an enzyme forwhich there are chromogenic substrates; a luciferase (lux) gene (Ow etal., Science. 234:856-859.1986), which allows for bioluminescencedetection; or an aequorin gene (Prasher et al., Biochem. Biophys. Res.Comm. 126:1259-1268 (1985)), which may be employed in calcium-sensitivebioluminescence detection, or a green or yellow fluorescent protein gene(Niedz et al., Plant Cell Reports. 14:403 (1995)).

Another screenable marker contemplated for use is firefly luciferase,encoded by the lux gene. The presence of the lux gene in transformedcells may be detected using, for example, X-ray film, scintillationcounting, fluorescent spectrophotometry, low-light video cameras, photoncounting cameras or multiwell luminometry. It is also envisioned thatthis system may be developed for population screening forbioluminescence, such as on tissue culture plates, or even for wholeplant screening.

Other Optional Sequences: An expression cassette of the invention canalso include plasmid DNA. Plasmid vectors include additional DNAsequences that provide for easy selection, amplification, andtransformation of the expression cassette in prokaryotic and eukaryoticcells, e.g., pUC-derived vectors such as pUC8, pUC9, pUC18, pUC19,pUC23, pUCl 19, and pUC120, pSK-derived vectors, pGEM-derived vectors,pSP-derived vectors, or pBS-derived vectors. The additional DNAsequences can include origins of replication to provide for autonomousreplication of the vector, additional selectable marker genes, forexample, encoding antibiotic or herbicide resistance, unique multiplecloning sites providing for multiple sites to insert DNA sequences orgenes encoded in the expression cassette and sequences that enhancetransformation of prokaryotic and eukaryotic cells.

Another vector that is useful for expression in both plant andprokaryotic cells is the binary Ti plasmid (as disclosed in Schilperoortet al.. U.S. Pat. No. 4,940,838) as exemplified by vector pGA582. Thisbinary Ti plasmid vector has been previously characterized by An(Methods in Enzymology. 153:292 (1987)) and is available from Dr. An.This binary Ti vector can be replicated in prokaryotic bacteria such asE. coli and Agrobacterium. The Agrobacterium plasmid vectors can be usedto transfer the expression cassette to dicot plant cells, and undercertain conditions to monocot cells, such as rice cells. The binary Tivectors can include the nopaline T DNA right and left borders to providefor efficient plant cell transformation, a selectable marker gene,unique multiple cloning sites in the T border regions, the colE1replication of origin and a wide host range replicon. The binary Tivectors carrying an expression cassette of the invention can be used totransform both prokaryotic and eukaryotic cells but is usually used totransform dicot plant cells.

DNA Delivery of the DNA Molecules into Host Cells: Methods describedherein can include introducing nucleic acids encoding enzymes, such as apreselected cDNA encoding the selected enzyme, into a recipient cell tocreate a transformed cell. In some instances, the frequency ofoccurrence of cells taking up exogenous (foreign) DNA may be low.Moreover, it is most likely that not all recipient cells receiving DNAsegments or sequences will result in a transformed cell wherein the DNAis stably integrated into the plant genome and/or expressed. Somerecipient cells may show only initial and transient gene expression.However, certain cells from virtually any dicot or monocot species maybe stably transformed, and these cells regenerated into transgenicplants, through the application of the techniques disclosed herein.

Another aspect of the invention is a plant that can produce terpenes,diterpenes sesquiterpenes, terpenoids, and combinations thereof, whereinthe plant has introduced nucleic acid sequence(s) encoding one or moreenzymes. The plant can be a monocotyledon or a dicotyledon. Anotheraspect of the invention includes plant cells (e.g., embryonic cells orother cell lines) that can regenerate fertile transgenic plants and/orseeds. The cells can be derived from either monocotyledons ordicotyledons. In some embodiments, the plant or cell is a monocotyledonplant or cell. In some embodiments, the plant or cell is a dicotyledonplant or cell. For example, the plant or cell can be a tobacco plant orcell. The cell(s) may be in a suspension cell culture or may be in anintact plant part, such as an immature embryo, or in a specialized planttissue, such as callus, such as Type I or Type II callus.

Transformation of plant cells can be conducted by any one of a number ofmethods available in the art. Examples are: Transformation by direct DNAtransfer into plant cells by electroporation (U.S. Pat. Nos. 5,384,253and 5,472,869, Dekeyser et al., The Plant Cell. 2:591-602 (1990));direct DNA transfer to plant cells by PEG precipitation (Hayashimoto etal., Plant Physiol. 93:857-863 (1990)): direct DNA transfer to plantcells by microprojectile bombardment (McCabe et al., Bio/Technology.6:923-926 (1988); Gordon-Kamm et al., The Plant Cell. 2:603-618 (1990);U.S. Pat. Nos. 5,489,520; 5,538,877; and 5,538,880) and DNA transfer toplant cells via infection with Agrobacterium. Methods such asmicroprojectile bombardment or electroporation can be carried out with“naked” DNA where the expression cassette may be simply carried on anyE. coli-derived plasmid cloning vector. In the case of viral vectors, itis desirable that the system retain replication functions, but lack thefunctions for disease induction.

One method for dicot transformation, for example, involves infection ofplant cells with Agrobacterium tumefaciens using the leaf-disk protocol(Horsch et al., Science 227:1229-1231(1985). Methods for transformationof monocotyledonous plants utilizing Agrobacterium tumefaciens have beendescribed by Hiei et al. (European Patent 0 604 662, 1994) and Saito etal. (European Patent 0 672 752, 1995).

Monocot cells such as various grasses or dicot cells such as tobacco canbe transformed via microprojectile bombardment of embryogenic callustissue or immature embryos, or by electroporation following partialenzymatic degradation of the cell wall with a pectinase-containingenzyme (U.S. Pat. Nos. 5,384,253; and 5,472,869). For example,embryogenic cell lines derived from immature embryos can be transformedby accelerated particle treatment as described by Gordon-Kamm et al.(The Plant Cell. 2:603-618 (1990)) or U.S. Pat. Nos. 5,489,520;5,538,877 and 5,538,880, cited above. Excised immature embryos can alsobe used as the target for transformation prior to tissue cultureinduction, selection and regeneration as described in U.S. applicationSer. No. 08/112,245 and PCT publication WO 95/06128.

The choice of plant tissue source for transformation may depend on thenature of the host plant and the transformation protocol. Useful tissuesources include callus, suspensions culture cells, protoplasts, leafsegments, stem segments, tassels, pollen, embryos, hypocotyls, tubersegments, meristematic regions, and the like. The tissue source isselected and transformed so that it retains the ability to regeneratewhole, fertile plants following transformation, i.e., containstotipotent cells.

The transformation is carried out under conditions directed to the planttissue of choice. The plant cells or tissue are exposed to the DNA orRNA encoding enzymes for an effective period of time. This may rangefrom a less than one second pulse of electricity for electroporation toa 2-day to 3-day co-cultivation in the presence of plasmid-bearingAgrobacterium cells. Buffers and media used will also vary with theplant tissue source and transformation protocol. Many transformationprotocols employ a feeder layer of suspended culture cells (tobacco, forexample) on the surface of solid media plates, separated by a sterilefilter paper disk from the plant cells or tissues being transformed.

Electroporation: Where one wishes to introduce DNA by means ofelectroporation, it is contemplated that the method of Krzyzek et al.(U.S. Pat. No. 5,384,253) may be advantageous. In this method, certaincell wall-degrading enzymes, such as pectin-degrading enzymes, areemployed to render the target recipient cells more susceptible totransformation by electroporation than untreated cells. Alternatively,recipient cells can be made more susceptible to transformation, bymechanical wounding.

To effect transformation by electroporation, one may employ eitherfriable tissues such as a suspension cell cultures, or embryogeniccallus, or alternatively, one may transform immature embryos or otherorganized tissues directly. The cell walls of the preselected cells ororgans can be partially degraded by exposing them to pectin-degradingenzymes (pectinases or pectolyases) or mechanically wounding them in acontrolled manner. Such cells would then be receptive to DNA uptake byelectroporation, which may be carried out at this stage, and transformedcells then identified by a suitable selection or screening protocoldependent on the nature of the newly incorporated DNA.

Microprojectile Bombardment: A further advantageous method fordelivering transforming DNA segments to plant cells is microprojectilebombardment. In this method, microparticles may be coated with DNA anddelivered into cells by a propelling force. Exemplary particles includethose comprised of tungsten, gold, platinum, and the like.

It is contemplated that in some instances DNA precipitation onto metalparticles would not be necessary for DNA delivery to a recipient cellusing microprojectile bombardment. In an illustrative embodiment,non-embryogenic BMS cells were bombarded with intact cells of thebacteria E. coli or Agrobacterium tumefaciens containing plasmids witheither the β-glucoronidase or bar gene engineered for expression inselected plant cells. Bacteria were inactivated by ethanol dehydrationprior to bombardment. A low level of transient expression of thesI-glucoronidase gene was observed 24-48 hours following DNA delivery.In addition, stable transformants containing the bar gene were recoveredfollowing bombardment with either E. coli or Agrobacterium tumefacienscells. It is contemplated that particles may contain DNA rather than becoated with DNA. Hence it is proposed that particles may increase thelevel of DNA delivery but are not, in and of themselves, necessary tointroduce DNA into plant cells.

An advantage of microprojectile bombardment, in addition to being aneffective means of reproducibly stably transforming monocots,microprojectile bombardment does not require the isolation ofprotoplasts (Christou et al., PNAS 84:3962-3966 (1987)), the formationof partially degraded cells, and no susceptibility to Agrobacteriuminfection is required. An illustrative embodiment of a method fordelivering DNA into maize cells by acceleration is a Biolistics ParticleDelivery System, which can be used to propel particles coated with DNAor cells through a screen, such as a stainless steel or Nytex screen,onto a filter surface covered with maize cells cultured in suspension(Gordon-Kamm et al., The Plant Cell. 2:603-618 (1990)). The screendisperses the particles so that they are not delivered to the recipientcells in large aggregates. It is believed that a screen interveningbetween the projectile apparatus and the cells to be bombarded reducesthe size of projectile aggregate and may contribute to a higherfrequency of transformation, by reducing the damage inflicted onrecipient cells by an aggregated projectile.

For bombardment, cells in suspension are preferably concentrated onfilters or solid culture medium. Alternatively, immature embryos orother target cells may be arranged on solid culture medium. The cells tobe bombarded are positioned at an appropriate distance below themicroprojectile stopping plate. If desired, one or more screens are alsopositioned between the acceleration device and the cells to bebombarded. Through the use of techniques set forth herein, one mayobtain up to 1000 or more foci of cells transiently expressing a markergene. The number of cells in a focus which express the exogenous geneproduct 48 hours post-bombardment often range from about 1 to 10 andaverage about 1 to 3.

In bombardment transformation, one may optimize the prebombardmentculturing conditions and the bombardment parameters to yield the maximumnumbers of stable transformants. Both the physical and biologicalparameters for bombardment can influence transformation frequency.Physical factors are those that involve manipulating theDNA/microprojectile precipitate or those that affect the path andvelocity of either the macro- or microprojectiles. Biological factorsinclude all steps involved in manipulation of cells before andimmediately after bombardment, the osmotic adjustment of target cells tohelp alleviate the trauma associated with the bombardment, and also thenature of the transforming DNA, such as linearized DNA or intactsupercoiled plasmid DNA.

One may wish to adjust various bombardment parameters in small scalestudies to fully optimize the conditions and/or to adjust physicalparameters such as gap distance, flight distance, tissue distance, andhelium pressure. One may also minimize the trauma reduction factors(TRFs) by modifying conditions which influence the physiological stateof the recipient cells and which may therefore, influence transformationand integration efficiencies. For example, the osmotic state, tissuehydration and the subculture stage or cell cycle of the recipient cellsmay be adjusted for optimum transformation. Execution of such routineadjustments will be known to those of skill in the art.

Selection: An exemplary embodiment of methods for identifyingtransformed cells involves exposing the bombarded cultures to aselective agent, such as a metabolic inhibitor, an antibiotic, or thelike. Cells which have been transformed and have stably integrated amarker gene conferring resistance to the selective agent used, will growand divide in culture. Sensitive cells will not be amenable to furtherculturing.

To use the bar-bialaphos or the EPSPS-glyphosate selective system,bombarded tissue is cultured for about 0-28 days on nonselective mediumand subsequently transferred to medium containing from about 1-3 mg/lbialaphos or about 1-3 mM glyphosate, as appropriate. While ranges ofabout 1-3 mg/l bialaphos or about 1-3 mM glyphosate can be employed, itis proposed that ranges of at least about 0.1-50 mg/I bialaphos or atleast about 0.1-50 mM glyphosate will find utility in the practice ofthe invention. Tissue can be placed on any porous, inert, solid orsemi-solid support for bombardment, including but not limited to filtersand solid culture medium. Bialaphos and glyphosate are provided asexamples of agents suitable for selection of transformants, but thetechnique of this invention is not limited to them.

The enzyme luciferase is also useful as a screenable marker in thecontext of the present invention. In the presence of the substrateluciferin, cells expressing luciferase emit light which can be detectedon photographic or X-ray film, in a luminometer (or liquid scintillationcounter), by devices that enhance night vision, or by a highly lightsensitive video camera, such as a photon counting camera. All of theseassays are nondestructive and transformed cells may be cultured furtherfollowing identification. The photon counting camera is especiallyvaluable as it allows one to identify specific cells or groups of cellswhich are expressing luciferase and manipulate those in real time.

It is further contemplated that combinations of screenable andselectable markers may be useful for identification of transformedcells. For example, selection with a growth inhibiting compound, such asbialaphos or glyphosate at concentrations that provide 100% inhibitionfollowed by screening of growing tissue for expression of a screenablemarker gene such as luciferase would allow one to recover transformantsfrom cell or tissue types that are not amenable to selection alone.

Regeneration and Seed Production: Cells that survive the exposure to theselective agent, or cells that have been scored positive in a screeningassay, are cultured in media that supports regeneration of plants. Oneexample of a growth regulator that can be used for such purposes isdicamba or 2,4-D. However, other growth regulators may be employed,including NAA, NAA+2,4-D or perhaps even picloram. Media improvement inthese and like ways can facilitate the growth of cells at specificdevelopmental stages. Tissue can be maintained on a basic media withgrowth regulators until sufficient tissue is available to begin plantregeneration efforts, or following repeated rounds of manual selection,until the morphology of the tissue is suitable for regeneration, atleast two weeks, then transferred to media conducive to maturation ofembryoids. Cultures are typically transferred every two weeks on thismedium. Shoot development signals the time to transfer to medium lackinggrowth regulators.

The transformed cells, identified by selection or screening and culturedin an appropriate medium that supports regeneration, can then be allowedto mature into plants. Developing plantlets are transferred to soillessplant growth mix, and hardened, e.g., in an environmentally controlledchamber at about 85% relative humidity, about 600 ppm CO₂, and at about25-250 microeinsteins/sec·m² of light. Plants can be matured either in agrowth chamber or greenhouse. Plants are regenerated from about 6 weeksto 10 months after a transformant is identified, depending on theinitial tissue. During regeneration, cells are grown on solid media intissue culture vessels. Illustrative embodiments of such vessels arepetri dishes and Plant Con™. Regenerating plants can be grown at about19° C. to 28° C. After the regenerating plants have reached the stage ofshoot and root development, they may be transferred to a greenhouse forfurther growth and testing.

Mature plants are then obtained from cell lines that are known toexpress the trait. In some embodiments, the regenerated plants areself-pollinated. In addition, pollen obtained from the regeneratedplants can be crossed to seed grown plants of agronomically importantinbred lines. In some cases, pollen from plants of these inbred lines isused to pollinate regenerated plants. The trait is geneticallycharacterized by evaluating the segregation of the trait in first andlater generation progeny. The heritability and expression in plants oftraits selected in tissue culture are of particular importance if thetraits are to be commercially useful.

Regenerated plants can be repeatedly crossed to inbred plants tointrogress the nucleic acids encoding an enzyme into the genome of theinbred plants. This process is referred to as backcross conversion. Whena sufficient number of crosses to the recurrent inbred parent have beencompleted in order to produce a product of the backcross conversionprocess that is substantially isogenic with the recurrent inbred parentexcept for the presence of the introduced nucleic acids, the plant isself-pollinated at least once in order to produce a homozygous backcrossconverted inbred containing the nucleic acids encoding the enzyme(s).Progeny of these plants are true breeding.

Alternatively, seed from transformed plants regenerated from transformedtissue cultures is grown in the field and self-pollinated to generatetrue breeding plants.

Seed from the fertile transgenic plants can then be evaluated for thepresence and/or expression of the enzyme(s). Transgenic plant and/orseed tissue can be analyzed for enzyme expression using methods such asSDS polyacrylamide gel electrophoresis, Western blot, liquidchromatography (e.g., HPLC) or other means of detecting an enzymeproduct (e.g., a terpene, diterpene, sesquiterpene, terpenoid, or acombination thereof).

Once a transgenic seed or plant expressing the enzyme(s) and producingone or more terpenes, diterpenes, sesquiterpenes, and/or terpenoids inthe plant is identified, the seed can be used to develop true breedingplants. The true breeding plants are used to develop a line of plantsexpressing terpenes, diterpenes, sesquiterpene, and/or terpenoids invarious plant tissues (e.g., in leaves, bracts, and/or trichomes) whilestill maintaining other desirable functional agronomic traits. Addingthe trait of terpene, diterpene, sesquiterpene, and/or terpenoidproduction can be accomplished by back-crossing with selected desirablefunctional agronomic trait(s) and with plants that do not exhibit suchtraits and studying the pattern of inheritance in segregatinggenerations. Those plants expressing the target trait(s) in a dominantfashion are preferably selected. Back-crossing is carried out bycrossing the original fertile transgenic plants with a plant from aninbred line exhibiting desirable functional agronomic characteristicswhile not necessarily expressing the trait of terpene, diterpene,sesquiterpenes, and/or terpenoid production in the plant. The resultingprogeny can then be crossed back to the parent that expresses theterpenes, diterpenes, sesquiterpenes, and/or terpenoids. The progenyfrom this cross will also segregate so that some of the progeny carrythe trait and some do not. This back-crossing is repeated until the goalof acquiring an inbred line with the desirable functional agronomictraits, and with production of terpenes, diterpenes, sesquiterpenes,and/or terpenoids within various tissues of the plant is achieved. Theenzymes can be expressed in a dominant fashion.

Subsequent to back-crossing, the new transgenic plants can be evaluatedfor synthesis of terpenes, diterpenes, sesquiterpenes, and/or terpenoidsin selected plant lines. This can be done, for example, by gaschromatography, mass spectroscopy, or NMR analysis of whole plant cellwalls (Kim, H., and Ralph, J. Solution-state 2D NMR of ball-milled plantcell wall gels in DMSO-d₆/pyridine-d₅. (2010) Org. Biomol. Chem. 8(3),576-591; Yelle, D. J., Ralph, J., and Frihart, C. R. Characterization ofnon-derivatized plant cell walls using high-resolution solution-stateNMR spectroscopy. (2008) Magn. Reson. Chem. 46(6), 508-517; Kim, H.,Ralph, J., and Akiyama, T. Solution-state 2D NMR of Ball-milled PlantCell Wall Gels in DMSO-d6. (2008) BioEnergy Research 1(1), 56-66; Lu,F., and Ralph, J. Non-degradative dissolution and acetylation ofball-milled plant cell walls; high-resolution solution-state NMR. (2003)Plant J. 35(4), 535-544). The new transgenic plants can also beevaluated for a battery of functional agronomic characteristics such aslodging, yield, resistance to disease, resistance to insect pests,drought resistance, and/or herbicide resistance.

Determination of Stably Transformed Plant Tissues: To confirm thepresence of the nucleic acids encoding terpene synthesizing enzymes inthe regenerating plants, or seeds or progeny derived from theregenerated plant, a variety of assays may be performed. Such assaysinclude, for example, molecular biological assays, such as Southern andNorthern blotting and PCR; biochemical assays, such as detecting thepresence of enzyme products, for example, by enzyme assays, byimmunological assays (ELISAs and Western blots). Various plant parts canbe assayed, such as trichomes, leaves, bracts, seeds or roots. In somecases, the phenotype of the whole regenerated plant can be analyzed.

Whereas DNA analysis techniques may be conducted using DNA isolated fromany part of a plant, RNA may only be expressed in particular cells ortissue types and so RNA for analysis can be obtained from those tissues.PCR techniques may also be used for detection and quantification of RNAproduced from introduced nucleic acids. PCR can also be used to reversetranscribe RNA into DNA, using enzymes such as reverse transcriptase,and then this DNA can be amplified through the use of conventional PCRtechniques. Further information about the nature of the RNA product maybe obtained by Northern blotting. This technique will demonstrate thepresence of an RNA species and give information about the integrity ofthat RNA. The presence or absence of an RNA species can also bedetermined using dot or slot blot Northern hybridizations. Thesetechniques are modifications of Northern blotting and also demonstratethe presence or absence of an RNA species.

While Southern blotting may be used to detect the nucleic acid encodingthe enzyme(s) in question, it may not provide information as to whetherthe preselected DNA segment is being expressed. Expression may beevaluated by specifically identifying the protein products of theintroduced nucleic acids or evaluating the phenotypic changes broughtabout by their expression.

Assays for the production and identification of specific proteins maymake use of physical-chemical, structural, functional, or otherproperties of the proteins. Unique physical-chemical or structuralproperties allow the proteins to be separated and identified byelectrophoretic procedures, such as, native or denaturing gelelectrophoresis or isoelectric focusing, or by chromatographictechniques such as ion exchange, liquid chromatography or gel exclusionchromatography. The unique structures of individual proteins offeropportunities for use of specific antibodies to detect their presence informats such as an ELISA assay. Combinations of approaches may beemployed with even greater specificity such as Western blotting in whichantibodies are used to locate individual gene products that have beenseparated by electrophoretic techniques. Additional techniques may beemployed to absolutely confirm the identity of the enzyme such asevaluation by amino acid sequencing following purification. Otherprocedures may be additionally used.

The expression of a gene product can also be determined by evaluatingthe phenotypic results of its expression. These assays also may takemany forms including but not limited to analyzing changes in thechemical composition, morphology, or physiological properties of theplant. Chemical composition may be altered by expression of preselectedDNA segments encoding storage proteins which change amino acidcomposition and may be detected by amino acid analysis.

Hosts

Terpenes, including diterpenes and terpenoids, can be made in a varietyof host organisms either in vitro or in vivo. In some cases, the enzymesdescribed herein can be made in host cells, and those enzymes can beextracted from the host cells for use in vitro. As used herein, a “host”means a cell, tissue or organism capable of replication. The host canhave an expression cassette or expression vector that can include anucleic acid segment encoding an enzyme that is involved in thebiosynthesis of terpenes.

The term “host cell”, as used herein, refers to any prokaryotic oreukaryotic cell that can be transformed with an expression cassettes orvector carrying the nucleic acid segment encoding an enzyme that isinvolved in the biosynthesis of one or more terpenes. The host cellscan, for example, be a bacterial, insect, plant, fungal, or yeast cell.Expression cassettes encoding biosynthetic enzymes can be incorporatedor transferred into a host cell to facilitate manufacture of the enzymesdescribed herein or the terpene, diterpene, or terpenoid products ofthose enzymes. The host cells can be present in an organism. Forexample, the host cells can be present in a host such as bacteria orfungi.

For example, the enzymes, terpenes, diterpenes, and terpenoids can bemade in a variety of bacteria or fungal cells. The enzymes, terpenes,diterpenes, and terpenoids can be made and extracted from such cells.Enzymes can conveniently, for example, be produced in bacterial, insect,plant, or fungal (e.g., yeast) cells.

Examples of host cells, host tissues, host fungi, host seeds and plantsthat may be used for producing terpenes and terpenoids (e.g., byincorporation of nucleic acids and expression systems described herein).Examples include bacterial and fungal host cells. For example, theterpenes and terpenoids can be produced in bacteria as described inShukal et al. Metabolic Engineering 55: 170-178 (2019).

The terpenes can also be produced in plants, such as those useful forproduction of oils such as oilseeds, camelina, canola, castor bean,corn, flax, lupins, peanut, potatoes, safflower, soybean, sunflower,cottonseed, oil firewood trees, rapeseed, rutabaga, sorghum, walnut, andvarious nut species. Other types host cells, host tissues, host seedsand plants that can be used include fiber-containing plants, trees,flax, grains (maize, wheat, barley, oats, rice, sorghum, millet andrye), grasses (switchgrass, prairie grass, wheat grass, sudangrass,sorghum, straw-producing plants), softwood, hardwood and other woodyplants (e.g., poplar, pine, and eucalyptus), oil (oilseeds, camelina,canola, castor bean, lupins, potatoes, soybean, sunflower, cottonseed,oil firewood trees, rapeseed, rutabaga, sorghum), starch plants (wheat,potatoes, lupins, sunflower and cottonseed), and forage plants (alfalfa,clover and fescue). In some embodiments the plant is a gymnosperm.Examples of plants useful for pulp and paper production include mostpine species such as loblolly pine, Jack pine, Southern pine, Radiatapine, spruce, Douglas fir and others. Hardwoods that can be modified asdescribed herein include aspen, poplar, eucalyptus, and others. Plantsuseful for making biofuels and ethanol include corn, grasses (e.g.,miscanthus, switchgrass, and the like), as well as trees such as poplar,aspen, pine, oak, maple, walnut, rubber tree, willow, and the like.Plants useful for generating forage include legumes such as alfalfa, aswell as forage grasses such as bromegrass, and bluestem. In some cases,the plant is a Brassicaceae or other Solanaceae species. In someembodiments, the plant is not a species of Arabidopsis, for example, insome embodiments, the plant is not Arabidopsis thaliana.

Additional examples of hosts cells and host organisms include, withoutlimitation, tobacco cells such as Nicotiana benthamiana, Nicotianatabacum, Nicotiana rustica, Nicotiana excelsior, and Nicotianaexcelsiana cells; cells of the genus Escherichia such as the speciesEscherichia coli; cells of the genus Clostridium such as the speciesClostridium ljungdahlii, Clostridium autoethanogenum or Clostridiumkluyveri; cells of the genus Corynebacterium such as the speciesCorynebacterium glutamicun; cells of the genus Cupriavidus such as thespecies Cupriavidus necator or Cupriavidus metallidurans; cells of thegenus Pseudomonas such as the species Pseudomonas fluorescens.Pseudomonas putida or Pseudomonas oleavorans: cells of the genus Delftiasuch as the species Delftia acidovorans; cells of the genus Bacillussuch as the species Bacillus subtilis: cells of the genus Lactobacillussuch as the species Lactobacillus delbrueckii; or cells of the genusLactococcus such as the species Lactococcus lactis.

“Host cells” can further include, without limitation, those from yeastand other fungi, as well as, for example, insect cells. Examples ofsuitable eukaryotic host cells include yeasts and fungi from the genusAspergillus such as Aspergillus niger; from the genus Saccharomyces suchas Saccharomyces cerevisiae; from the genus Candida such as C.tropicalis, C. albicans, C. cloacae, C. guillermondii, C. intermedia, C.maltosa, C. parapsilosis, and C. zeylenoides; from the genus Pichia (orKomagataella) such as Pichia pastoris; from the genus Yarrowia such asYarrowia lipolytica; from the genus Issatchenkia such as Issathenkiaorientalis: from the genus Debaryomyces such as Debaryomyces hansenii;from the genus Arxula such as Arxula adenoinivorans; or from the genusKluyveromyces such as Kluyveromyces lactis or from the genera Erophiala,Mucor, Trichoderma, Cladosporium, Phanerochaete, Cladophialophora,Paecilomyces, Scedosporium, and Ophiostoma.

In some cases, the host cells can have organelles that facilitatemanufacture or storage of the terpenes, diterpenes, and terpenoids. Suchorganelles can include lipid droplets, smooth endoplasmic reticulum,plastids, trichomes, vacuoles, vesicles, plastids, and cellularmembranes. During and after production of the terpenes, diterpenes, andterpenoids these organelles can be isolated as a semi-pure source of theof the terpenes, diterpenes, sesquiterpenes, and terpenoids.

Manufacturing

Also described herein are methods of synthesizing a terpene that includeincubating a host cell that has any of the expression cassettes orexpression systems described herein. For example, the terpenes andterpenoids can be produced in bacteria as described in Shukal et al.Metabolic Engineering 55: 170-178 (2019), which is incorporated byreference herein in its entirety (and provided as Appendix B).

In addition, the application includes methods of synthesizing a terpenethat include cultivating a seed or plant that has any of the expressioncassettes or expression systems described herein to produce a planthaving terpenes in its tissues.

Such methods can also include isolation of the terpene(s) from the hostcells, or the plants having terpenes in its tissues.

The terpene that is synthesized and/or isolated can be viridiflorol.

Volatile organic compounds, including volatile terpenoids such asviridiflorol, mediate communication between plants and microbes (Wenkeet al., 2010). Plant terpenoids have been positively involved in thedevelopment of ectomycorrhizal associations with several plant speciesduring the pre-symbiotic phase (Fries et al., 1987: Menotta et al.,2004). Despite a couple of attempts (Crutcher et al., 2013), there is nodirect evidence that fungal terpenoids could play a similar role andfacilitate establishment of plant-endophytic interactions. Terpenoidsproduced by these fungi are most frequently implicated in defenseagainst antagonists (Minerdi et al., 2009) or in intraspecies andinterspecies recognition (Hynes et al., 2006). Colonization experiments,including experiments with knockout mutants, and antagonism assaysbetween S. indica and other microorganisms such as those describedherein offer a clearer image about the role of SiTPS and the producedcompounds in the establishment of S. indica-plant association and thedefense mechanisms of the endophyte.

The following Examples illustrate some of the experimental work involvedin developing the invention.

Example 1: Materials and Methods

This Example describes some of the materials and methods used in thedevelopment of the invention.

Fungal and Plant Material

Serendipita indica (DSM11827 isolate) was grown at 28° C. in liquidcomplete medium (CM) (Pham et al., 2004) supplemented with 2% glucose wv⁻¹ and shaking at 150 rpm, or on solid CM plates (supplemented with 2%glucose w v⁻¹ and 1.5% w v⁻¹ agar).

Tomato seeds (Solanum lycopersicum, cv. Moneymaker) were surfacesterilized with 70% ethanol for 1 min, 1% NaClO (v v⁻¹) for 10 min andrinsed with sterile MilliQ water. The seeds were germinated for 11 dayson sterile filter paper in a growth chamber (12 h day 22° C./12 h night18° C., 120 μE m⁻² s⁻¹ light intensity, 60% relative humidity). Plantinoculation with S. indica wild type and mutants was performed byincubating the tomato seedlings in 40 ml of fungal inoculum (300,000chlamydospores ml⁻¹) overnight, on a shaker (120 rpm) at roomtemperature. For the control treatment, sterile water was used insteadof the fungal chlamydospore suspension. After inoculation, the seedlingswere sown on Murashige-Skoog (MS) Basal medium (Sigma-Aldrich, USA)supplemented with 1.5% w v⁻¹ agar and grown in the same growth chamberuntil harvesting.

Discovery of SiTPS and Phylogenetic Analysis of Putative TerpeneSynthase

The genome of S. indica (available at the Joint Genome Institute-JGIwebsite, genome.jgi.doe.gov/Pirin1/Pirin1.home.html) indicated that S.indica possesses one gene (JGI mRNA:PIIN_06735), which based on theannotation belongs to the Terpene_synth_C (μF03936) family (Zuccaro etal., 2011). This putative terpene synthase gene was named SiTPS and thepredicted amino acid sequence (JGI Protein Id: 77541) was used in aBLASTp search in order to identify similar proteins. A selection offungal and bacterial proteins were aligned with SiTPS sequence using theonline tool GUIDANCE2 Server (see website at guidance.tau.ac.il/ver2/)(MAFFT). The alignment file was imported in MEGA7 (Kumar et al., 2016)and a maximum-likelihood phylogenetic tree (bootstrap of 100) wasconstructed using the default settings (FIG. 2 ).

SiTPS was also aligned with selected functionally characterizedBasidiomycota and Ascomycota STSs, including the Cop1-6 (Agger et al.,2009; Lopez-Gallego et al., 2010a,b) and Omp1-10 (Wawrzyn et al., 2012b)using again the GUIDANCE2 Server (MAFFT). A Neighbour-joiningphylogenetic tree was constructed in MEGAX (Kumar et al., 2018)(bootstrap of 100) using the default settings.

Heterologous Expression in Escherichiacoli and In Vitro Characterizationof SiTPS

The coding region of SiTPS (PIIN_06735) was amplified from fungal cDNAusing the primers shown in Table 2.

TABLE 2 Primers SEO ID NO: Name Sequence 5’-3’ Used for 11 SiTPSAGAAGGAGATATA In-fusion ® cloning inf-F CCATGCCATCTGTCT pET_SiTPSCTCCTGCCAC 12 SiTPS GGTGGTGGTGCTC In-fusion ® cloning inf-RGAACGCAACGGGA pET_SiTPS GCCACTGGG 13 TPS CTCCAAAAACAGTin-fusion ® cloning ovinf-F CGATGCCATCTGTC K167_SiTPS_His TCTCCTGC 14TPS TAGATATCGTAG In-fusion ® cloning Ovinf TTTGCAACGGGAGC K167_SiTPS_HisHA-R CACTGGGGT 15 HD1.1-F CGATACCTACC S. indica CGCCTACAAmating type locus HD 1.1 (PIN_09915) 16 HD1.1-R CTTTTTAAGC S. indicaGGTGCTGGAG mating type locus HD 1.1 (PIN_09915) 17 HD2.1-R ATGAGTACGAS. indica TTGCCCAAGG mating type locus HD 2.1 (PIN_09916) 18 HD2.1-FTCGTCTCGT S. indica AGGCGACTTTT mating type locus HD 2.1 (PIN_09916) 19HD1.2-R AGATATCCGG S. indica AGGCGAGTTT mating type locus HD 1.2(PIN_09977) 20 HD1.2-F CCTGAATCTG S. indica CTGTTCGTCAmating type locus HD 1.2 (PIN_09977) 21 HD2.2-F ACATCTGGCT S. indicaCCCATTTACG mating type locus HD 2.2 (PIN_09978) 22 HD2.2-R GTTGAGCTTTGS. indica GCTCGTTTC mating type locus HD 2.2 (PIN_09978) 23 SiITS-FCAACACATGTG S. indica ITS CACGTCGAT estimation of fungal biomass 24SiITS-R CCAATGTGCA S. indica ITS TTCAGAACGA estimation of fungal biomass25 SlTUB-F AACCTCCATTC Solanum lycopersicum AGGAGATGTTTβ-tubulin estimation of plant biomass 26 SlTUB-R TGCTGTAGCATSolanum lycopersicum CCTGGTATT β-tubulin estimation of plant biomass

The coding region of SiTPS was then cloned into pET28b+ vector (Novagen)using the In-Fusion® HD Cloning Kit (Takara Bio, USA), according tomanufacturer's instructions. The plasmid containing SiTPS, namedpET_SiTPS, was transformed into E. coli C41 OverExpress™ cells (Lucigen,USA). Heterologous expression from the pET_SiTPS vector and proteinpurification was performed as described in Johnson et al. (2018).Briefly, 500 μl of an overnight culture (5 ml LB medium broth containing50 μg ml⁻¹ kanamycin) was used to inoculate 50 ml of production medium(Terrific Broth medium [pH 7.0] containing 50 μg ml⁻¹ kanamycin).Cultures were grown at 37° C. in a shaking incubator (180 rpm). When theculture OD₆₀₀ reached 0.6, 100 μl of IPTG (Isopropylβ-D-1-thiogalactopyranoside, 0.2 mM) were added to induce expression.Protein expression proceeded overnight at 16° C. in a shaking incubator(180 rpm). The following day, cells were harvested by centrifugation(4500 rpm) at 4° C. for 20 min and lysed using the CelLytic B Cell LysisReagent (Sigma-Aldrich. USA) supplemented with 0.1 mg ml⁻¹ lysozyme, 10μl ml⁻¹ protease inhibitor cocktail (Sigma), 0.2 mg ml⁻¹ benzoase, 25 mMimidazole, 500 mM NaCl and 5% v v⁻¹ glycerol. The cell lysate was usedfurther for protein purification, using the His SpinTrap Kit (GEHealthcare, USA). Protein desalting was done with the PD MiniTrap G-25desalting columns (GE Healthcare, USA) according to the manufacturer'sinstructions and 600 μl of desalting buffer (20 mM HEPES [pH 7.2], 1 mMMgCl₂, 350 mM NaCl, 5 mM DTT, and 5% v v⁻¹ glycerol) were used to elutethe purified protein.

The in vitro terpene synthase assay was performed in a 500 μl reactionthat contained 5 μg substrate (GPP, 2E,6E-FPP, or GGPP [CaymanChemicals, USA]), 100 μg purified enzyme, 10 mM MgCl₂, 100 mM KCl, 5 mMDTT, and 10% v v⁻¹ glycerol in 50 mM HEPES (pH 7.2). The reaction wasoverlaid with 500 μl n-hexane. Reactions were carried out at 30° C. for1 hour, followed by vortexing to extract the products into the organicphase. Layers were separated by centrifugation, and hexane was removedfor GC-MS analysis.

In Vivo Characterization of SiTPS

For the characterization of SiTPS in vivo, two more plasmids apart frompET_SiTPS, were used. The in vivo system was established by modifyingthe diterpene production system previously developed by Morrone et al.(2010). The GPP synthase gene was removed from the pGG vector (Cyr etal., 2007) and replaced by a Gallus gallus FPP synthase gene(FPPS-Genbank XM_01529864). In detail, the G. gallus FPPS wassynthesized (Integrated DNA Technologies, USA) and cloned intoNdeI-digested and XhoI-digested pACYCDuet vector (Novagen) using theIn-Fusion® HD Cloning Kit (Takara Bio, USA). The resulted plasmid wasnamed pFF, and together with pIRS, a plasmid containing three upstreamgenes of the MEP pathway (Morrone et al., 2010), was introduced to E.coli C41 OverExpress™ cells (Lucigen, USA) to create a farnesyldiphosphate-producing strain. This strain was transformed with pET_SiTPSfor the in vivo characterization of SiTPS. Transformed E. coli cellswere grown on LB agar plates containing kanamycin (25 μg ml⁻¹),chloramphenicol (20 μg ml⁻¹), and streptomycin (25 μg ml⁻¹), and werefurther screened for the presence of all the plasmids using colony PCR.A single PCR-positive clone was grown in 50 ml Terrific Broth medium (pH7.0), with the antibiotics mentioned above. The culture was grown at 37°C. to reach an OD₆₀₀ of 0.6, after which the temperature dropped to 16°C. for 1 hour before expression was induced with 1 mM IPTG. The culturewas also supplemented with 40 mM pyruvate, 1 mM MgCl₂ and was grown foradditional 72 hours. Produced compounds were extracted in 50 ml ofn-hexane. After separation, the organic phase was concentrated under N₂and analyzed by GC-MS.

Gas Chromatography-Mas Spectrometry (GC-MS) Analysis

GC-MS analysis was performed as described by Johnson et al. (2019) on anAgilent 7890A GC with an Agilent VF-5 ms column (30 m×250 μm×0.25 μm,with 10m EZ-Guard) and an Agilent 5975C detector. The inlet was set to250° C. splitless injection of 1 μl, helium carrier gas with column flowof 1 ml min⁻¹. The detector was activated after a three-minute solventdelay. The oven temperature ramp started at 80° C. hold for 1 min,increase 40° C. min⁻¹ to 130° C., increase 10° C. min⁻¹ to 250° C.,increase 100° C. min⁻¹ to 325° C. hold 3 min. Obtained spectra werecompared with NIST17 Mass Spectral Database. Analytical standards ofviridiflorol (CAS 552-02-3) and ledol (CAS 577-27-5) were purchased fromSigma Aldrich (Cat No. 72999-10MG) and Santa Cruz (Cat No. sc-396548),respectively.

SiTPS Expression in Planta and in Vitro

Tomato seedlings were harvested 11 dpi (days post inoculation) and rootsfrom seven plants were pooled together in one biological replication.Before RNA extraction, the samples were freeze-dried overnight tofacilitate milling. Material from mature S. indica cultures, grown fortwo weeks on CM agar plates, was also harvested. Total RNA from root andfungal samples was extracted using the Spectrum™, Plant Total RNA Kit(Sigma-Aldrich, USA). cDNA was synthesized with the Revert First StrandcDNA Synthesis kit and an oligo-dT primer (Thermo Fisher Scientific,USA).

For the RT-qPCR, 600 ng of cDNA was used as a template in a 10 μlreaction, using 5 μl of the Brilliant III Ultra-Fast SYBR® Green(Agilent Technologies, USA) and SiTPS-specific primers (Table 2). EachRT-qPCR reaction was performed with three technical replications for thefour biological replications. RT-qPCR was performed using the AriaMxReal-Time PCR System-G8830A (Agilent Technologies, USA) with a programof 95° C. for 5 min, followed by 40 cycles of 95° C. for 30 sec, 60° C.for 1 min, and 72° C. for 1 min. A final dissociation step was performedto assess the quality of amplified products and the specificity of theprimers. Expression of SiTPS was normalized using S. indicaglyceraldehyde-3-phosphate dehydrogenase gene (GAPDH. GenBank:FJ810523.1) expression levels and calculated with the 2^(−ΔCt) method(Livak & Schmittgen, 2001; Schmittgen & Livak, 2008).

Construction of Overexpression Plasmids and P. indica PEG-MediatedTransformation

For creating a SiTPS-overexpressing mutant, SiTPS coding sequence wascloned into NheI and PmeI digested K167 vector (Wawra and Widmer,unpublished) using the In-Fusion® HD Cloning Kit (Takara Bio, USA),resulting to the plasmid K167_SiTPSov (FIG. 6 ). The empty vector namedK167_ev was also used for S. indica transformation as acontrol-treatment.

S. indica protoplasts were isolated and transformed with K167_SiTPSovand K167_ev through polyethylene glycol (PEG)-mediated transformationaccording to Hilbert et al., 2012. Young mycelium from a 7-day-old S.indica culture was harvested, crushed and left to regenerate for threefurther days. The regenerated mycelium was treated with 20 ml solution(1.33 M sorbitol, 50 mM CaCl2 and 20 mM MES) containing 0.02 g ml⁻¹lysing enzyme from Trichoderma harzianum (Sigma-Aldrich, USA) at 32° C.After 2 h, protoplasts were harvested and transformed with 7-10 μg oflinearized and purified plasmid in the presence of 40% PEG 3350 andheparin (15 mg ml⁻¹). Protoplast regeneration was done using plates withtwo layers of malt yeast peptone (MYP) agar supplemented with 0.3 Msucrose (0.7% w v⁻¹ malt extract, 0.1% w v⁻¹ peptone, 0.05% w v⁻¹ yeastextract). The bottom medium contained 1.2% agar and hygromycin B (80 μgml⁻¹). The top medium (0.6% agar and no antibiotics) was mixed with thetransformation mixture and then quickly poured on to the solidifiedbottom medium. The plates were incubated at 28° C. After 10-14 days,regenerated colonies were transferred to CM plates supplemented with 80μg/ml hygromycin B and checked for carrying either K167_SiTPS orK167_EV. S. indica mutants used for further studies were selected basedon their growth and their mating type.

Quantification of Root Colonization by S. indica and Mutants with qPCR

Genomic DNA was extracted from root samples of inoculated tomato plants2- and 11-days post inoculation (dpi) using the DNeasy@ Plant Mini Kit(Qiagen, Germany). Roots from 8 plants were pooled in one biologicalreplication and 3 biological replications were included per time-point,per treatment. The qPCR was performed in the AriaMx Real-Time PCRSystem-G8830A (Agilent Technologies, USA) using a cycling program of 95°C. for 5 min, 30 cycles of 95° C. for 30 sec, 62° C. for 1 min, and 72°C. for 1 min. A final dissociation step was performed to assess thequality of amplified products and the specificity of the primers. In a10 μl reaction, 5 μl of the Brilliant III Ultra-Fast SYBR® Green(Agilent Technologies, USA), 1 μl of total DNA and 0.4 μM of plant- orfungus-specific primers were used (Table 2).

Fungal colonization was determined using the ratio of fungal to plantDNA amount. For the quantification of fungal DNA, a standard curve, madeusing serial dilutions of DNA from an S. indica pure culture andspecific primers (SiITS, GenBank: NR_119580.1), was used. Plant DNA wasquantified similarly, using a standard curve of plant DNA dilutions andspecific primers (SITUB, GenBank: DQ205342.1).

Example 2: Phylogenetic Analysis on SiTPS

The phylogenetic analysis using proteins with the highest sequencesimilarity to SiTPS (FIG. 2 ) showed that its closest relative is aterpenoid synthase, found in the orchid mycorrhizal fungus Serendipitavermifera (previously known as Sebacina vermifera) belonging to the samegenus. However, the activity of S. vermifera terpenoid synthase remainsunknown giving also no information about SiTPS function. Closely relatedto these two Sebacinales TPSs appeared to be a protein from the lichenCladonia uncialis, annotated as a putative pentalenene synthase(Bertrand et aL, 2018). No experimental evidence on the function of thisenzyme is available either.

The phylogenetic tree constructed from a selection of characterizedfungal STSs including Cop1-6 (Agger et aL, 2009; Lopez-Gallego et al.,2010a), Omp1-10, Fompi 1 (Wawrzyn et al., 2012b), Stehi11159379, 128017,25180, 64702, 73029 (Quin et al., 2015; Flynn & Schmidt-Dannert, 2018)and more, revealed that these enzymes fall in one of the four distinctSTS clades. SiTPS is a member of Clade I, which includes STSs thatcatalyze formation of sesquiterpenoids through an initial C1-C10 closureof 2E,6E-FPP.

The phylogenetic analysis on SiTPS-related amino acid sequences (FIG. 2) showed that the closer relative to SiTPS is the terpenoid synthasefrom S. vermifera, also a member of the Sebacinales order (Ray et aL,2018). However, since the gene from S. vermifera has not beenfunctionally characterized, it is not clear whether clustering of thetwo terpenoid synthases occurred due to taxonomical reasons or similarfunctions.

In addition, this phylogenetic analysis indicates that a clearseparation exists between fungal and bacterial sequences. The fungalcluster included proteins only from species of the Basidiomycota phylum,namely plant-symbionts e.g. S. vermifera and Tulasnella calospora(orchid symbionts) or decomposers of plant material [wood-rotting(Grifola frondosa, Gloeophyllum trabeum) and saprophytic fungi(Rhizoctonia solani). However, one of the terpenoid synthases(AUW30846.1) found in the fungal cluster, and actually closely-relatedto the Sebacinales cluster, belongs to the lichen Cladonia uncialis.

Lichens comprise symbiotic events between an autotroph organism (algaeor cyanobacteria) and a fungal partner. Lichenized fungi arephylogenetically widespread in the two phyla, Ascomycota andBasidiomycota (Yuan, 2005). However, the genus Cladonia includes onlyAscomycota lichenized fungi (Wang et al., 2011), meaning that AUW30846.1is the only representative of the Ascomycota phylum in the fungalcluster (FIG. 2 ). This indicates that similarity between the lichen TPSand the Sebacinales TPSs is not due to taxonomical proximity between theorganisms, but functional convergence.

Recently, the fungal symbiont of C. uncialis was isolated and its genomewas sequenced, revealing 48 secondary metabolite biosynthetic clustersand a vast potential for production of natural products. However, C.uncialis is known to produce only usnic acid, indicating that most ofthese gene clusters remain inactive under lab-culturing conditions.Among these clusters, nine terpene synthases were identified (Cu-terp-1to 9) and the gene product of Cu-terp-1 was the only one closely relatedto SiTPS (AUW30846.1). Based on sequence similarity to GenBankcharacterized genes, Cu-terp-I was proposed to be a pentalenene synthase(Bertrand et al., 2018). However, its function has not been validatedexperimentally.

A sequence for the Cladonia uncialis pentalenene synthase with accessionnumber AUW30846.1 is shown below as SEQ ID NO:5.

  1 MCAEWITVLF VWDDLLDVPI DSDLVSDEQG TREINRVMSC  41ILTQPENFEP MVTQPVTGAL HSFWTQFCAT SSPNMQKRFV  81EAVLKYAEGA AKQVASRETR ALPSIKDFIV NRQSASGVET 121LLALVEYCLQ IQVPDCAYYH PTLQQLRNSI NEIVSWSNDI 161YSFNKEQACG DHANLVVVVA IEKGIPVQSA ITYVSVMVQE 201AVKRYHENLK KIPKFDPRID ALVLKYVGGI ECVCTGLVSW 241HFKIDRYFGE NSSEVSNTLM VDLLPQEKNA LVSAHELQYD 281 QLPISTPQPK GSAMT

Example 3: SiTPS Encodes a Viridiflorol Synthase

SiTPS biochemical activity was elucidated using in vitm assays and an E.coli-based in vivo terpene production system. In the in vitro assays thepurified SiTPS, heterologously produced in E. coli cultures, was fedwith three terpene substrates (GPP, 2E,6E-FPP, GGPP). Hexane was used tooverlay the reactions and extract the produced compounds.

An E. coli-based in vivo system was used to validate results from the invitro assays. The E. coli cells were transformed with the plasmids pFFand pIRS, resulting to an FPP-producing system. Finally, theseFPP-producing strains were transformed with pET_SiTPS. After proteinexpression was completed, the E. coli cultures were extracted withhexane. GC-MS analysis on the hexane extract from the E. coli expressionsystem with all three plasmids (pIRS, pFF and pET_SiTPS) showed a numberof peaks that were absent in the extract derived from the FPP-producingcontrol (FIG. 3A). One peak, at 8.44 mins, was the most intense andconsidered to be the main product (FIG. 3A). The retention time and themass spectrum of this unique peak matched one of the products of the invitro assay with SiTPS and 2E,6E-FPP. A NIST17 database search showedthat the closest matches were the sesquiterpenes viridiflorol and ledol,but the retention time and the mass spectrum of SiTPS product wereidentical to that of the viridiflorol standard.

FIG. 3A shows chromatographic separation of the SiTPS products. Asillustrated in FIG. 3B, SiTPS showed activity only when it was incubatedwith FPP, validating its NCBI annotation as a “conventional”sesquiterpene synthase.

The in vitro and in vivo characterization assays showed that SiTPS isusing FPP to produce a mix of sesquiterpenoids with the main productidentified as viridiflorol (FIG. 3A). Viridiflorol is a sesquiterpenoidwith a 7/5/3 tricyclic scaffold and belongs to the group ofaromadendranes, terpenoids with a characteristic skeleton of a dimethylcyclopropane ring fused to a hydroazulene ring system (Gijsen et al.,1992).

One mechanism for the formation of viridiflorol starts with thecyclization of FPP by a ring closure of C1 and C10 (FIG.1B: see also,Hong & Tantillo (2011)). However, it is possible that FPP cyclisationoccurs either with the 1,10 closure of 2E,6E-FPP (route b) or itsisomer, (3R)-NPP(route a). More likely, SiTPS acts through route b,since phylogenetic analysis showed that it belongs to Clade I of STSs,which includes enzymes that form sesquiterpenes by preferably catalyzing1,10 cyclisation of 2E,6E-FPPcarbocation (FIG. 1B).

Viridiflorol has been detected in extracts of many plant species of theMyrtaceae (Padovan et al., 2010; Dreher et al., 2019) and the Lamiaceaefamilies (Medjahed et al., 2016; Ramirez et al., 2018). However, onlytwo proteins from the plant Melaleuca quinquenervia have been identifiedas viridiflorol synthases (Padovan et al., 2010). Viridiflorol has alsobeen encountered as a minor product in fungal extracts (Meshram et al.,2014; Wu et al., 2016), but there was no viridiflorol synthase gene of afungal origin reported until now. SiTPS is the first fungal viridiflorolsynthase identified and characterized experimentally.

Example 4: SiTPS is Induced Upon Root-Colonization

Expression of SiTPS was studied under two different growing conditions.Relative expression of SiTPS was quantified in fungal material from14-day-old S. indica colonies, grown on synthetic medium (here CM agar),and the characteristics thereof were compared to SiTPS expressingfungi-colonized tomato roots at 11 days post inoculation (FIG. 4 ).SiTPS gene expression was up-regulated 3-fold when S. indica was growingin planta instead of on CM agar plates. Hence, a moderate induction ofSiTPS expression was observed when the S. indica fungus is associatedwith plant tissues.

Example 5: SiTPS Implication in Colonization of Tomato Roots

Even though SiTPS was shown to express a functional viridiflorolsynthase in E. coli expression systems, no terpenoid was detected in S.indica cultures (data not shown). However. SiTPS was upregulated uponroot colonization (FIG. 4 ). These data indicated that SiTPS productsmay play a role in S. indica and plant root interactions.

To investigate if SiTPS can play a role in the ability of the fungus tocolonize plant roots, S. indica mutants that over-expressed SiTPS wereconstructed. S. indica protoplasts were transformed with a K167 plasmidcarrying a SiTPS coding sequence under the control of a strong promoter(FGB1 promoter-Fungal Glucan-Binding 1, PIIN_03211) (FIG. 6 ).

After a screening of the regenerative clones, one SiTPS-overexpressingtransformant (ovII) was selected and used in time-course colonizationexperiments to evaluate its ability to colonize plant roots. An S.indica-transformant carrying the empty K167 vector (controltreatment-evV) and the wild type S. indica were also used in the samecolonization studies (FIG. 7 ).

The ratio of fungal DNA to plant DNA was used as a measurement of thecolonization ability of each S. indica transformant. Colonization ratioswere estimated in colonized roots 2- and 11-days post-inoculation. Nodifference was observed in the colonization ability between theSiTPS-over-expressing transformants, the empty vector-carryingtransformants, and the wild type at the selected time-points. Nodifference was observed in colonization ability between S. indicatransformants and the wild type either (FIG. 5 ).

REFERENCES

-   Agger S, Lopez-Gallego F, Schmidt-Dannert C. 2009. Diversity of    sesquiterpene synthases in the basidiomycete Coprinus cinereus.    Molecular Microbiology 72: 1181-1195.-   Bajaj R, Huang Y, Gebrechristos S, Mikolajczyk B, Brown H, Prasad R,    Varma A, Bushley K E. 2018. Transcriptional responses of soybean    roots to colonization with the root endophytic fungus Piriformospora    indica reveals altered phenylpropanoid and secondary metabolism.    Scientific Reports 8: 10227.-   Bertrand R L, Abdel-Hameed M, Sorensen J L. 2018. Lichen    Biosynthetic Gene Clusters. Part 1. Genome Sequencing Reveals a Rich    Biosynthetic Potential. Journal of Natural Products 81: 723-731.-   Blackwell M. 2011. The Fungi: 1, 2, 3 . . . 5.1 million species?    American Journal of Botany 98: 426-438.-   Brock N L. Huss K, Tudzynski B, Dickschat J S. 2013. Genetic    Dissection of Sesquiterpene Biosynthesis by Fusarium fujikuroi.    ChemBioChem 14: 311-315.-   Caruthers J M, Kang 1, Rynkiewicz M J, Cane D E, Christianson    D W. 2000. Crystal Structure Determination of Aristolochene Synthase    from the Blue Cheese Mold, Penicillium roqueforti*. Journal of    Biological Chemistry 275: 25533-25539.-   Christianson D W. 2006. Structural Biology and Chemistry of the    Terpenoid Cyclases. Chemical Reviews 106: 3412-3442.-   Crutcher F K, Parich A, Schuhmacher R, Mukherjee P K, Zeilinger S,    Kenerley C M. 2013. A putative terpene cyclase, vir4, is responsible    for the biosynthesis of volatile terpene compounds in the biocontrol    fungus Trichoderma virens. Fungal Genetics and Biology 56: 67-77.-   Cyr A. Wilderman P R, Determan M, Peters R J. 2007. A Modular    Approach for Facile Biosynthesis of Labdane-Related Diterpenes.    Journal of the American Chemical Society 129: 6684-6685.-   Dreher D, Baldermann S, Schreiner M, Hause B. 2019. An arbuscular    mycorrhizal fungus and a root pathogen induce different volatiles    emitted by Medicago truncatula roots. Journal of Advanced Research:    0-5.-   Engels B, Heinig U, Grothe T, Stadler M, Jennewein S. 2011. Cloning    and Characterization of an Armillaria gallica cDNA Encoding    Protoilludene Synthase, Which Catalyzes the First Committed Step in    the Synthesis of Antimicrobial Melleolides. Journal of Biological    Chemistry 286: 6871-6878.-   Flynn C M, Broz K, Jonkers W, Schmidt-Dannert C, Kistler H C. 2019.    Expression of the Fusarium graminearum terpenome and involvement of    the endoplasmic reticulum-derived toxisome. Fungal Genetics and    Biology 124: 78-87.-   Flynn C M, Schmidt-Dannert C. 2018. Sesquiterpene    Synthase-3-Hydroxy-3-Methylglutaryl Coenzyme A Synthase Fusion    Protein Responsible for Hirsutene Biosynthesis in Stereum hirsutum    (C Vieille, Ed.). Applied and Environmental Microbiology 84: 1-18.-   Fries N, Serck-Hanssen K, Dimberg L H, Theander O. 1987. Abietic    acid, and activator of basidiospore germination in ectomycorrhizal    species of the genus Suillus (Boletaceae). Experimental Mycology 11:    360-363.-   Furtado F, Borges B, Teixeira T, Garces H, Almeida Junior L, Alves    F, Silva C, Fernandes Junior A. 2018. Chemical Composition and    Bioactivity of Essential Oil from Blepharocalyx salicifolius.    International Journal of Molecular Sciences 19: 33.-   Gershenzon J. Dudareva N. 2007. The function of terpene natural    products in the natural world. Nature Chemical Biology 3: 408-414.-   Gijsen HJM, Wijnberg JBPA, Stork G A, de Groot A, de Waard M A, van    Nistelrooy JGM. 1992. The synthesis of mono- and dihydroxy    aromadendrane sesquiterpenes, starting from natural    (+)-aromadendrene-III. Tetrahedron 48: 2465-2476.-   Hilbert M, Voll L M, Ding Y, Hofmann J, Sharma M, Zuccaro A. 2012.    Indole derivative production by the root endophyte Piriformospora    indica is not required for growth promotion but for biotrophic    colonization of barley roots. New Phytologist 196: 520-534.-   Hohn T M, Beremand P D. 1989. Isolation and nucleotide sequence of a    sesquiterpene cyclase gene from the trichothecene-producing fungus    Fusarium sporotrichioides. Gene 79: 131-138.-   Hong Y J, Tantillo D J. 2011. How Many Secondary Carbocations Are    Involved in the Biosynthesis of Avermitilol? Organic Letters 13:    1294-1297.-   Hynes J, Müller C T, Jones T H, Boddy L. 2006. Changes in Volatile    Production During the Course of Fungal Mycelial Interactions Between    Hypholoma fasciculare and Resinicium bicolor. Journal of Chemical    Ecology 33: 43-57.-   Jacobs S, Zechmann B, Molitor A. Trujillo M, Petutschnig E, Lipka V,    Kogel K-H, Schafer P. 2011. Broad-Spectrum Suppression of Innate    Immunity Is Required for Colonization of Arabidopsis Roots by the    Fungus Piriformospora indica. PLANT PHYSIOLOGY 156: 726-740.-   Johnson S R, Bhat W W, Bibik J, Turmo A, Hamberger B, Hamberger B.    2019a. A database-driven approach identifies additional diterpene    synthase activities in the mint family (Lamiaceae). Journal of    Biological Chemistry 294: 1349-1362.-   Johnson S R, Bhat W W, Sadre R, Miller G P, Garcia A S, Hamberger B.    2019b.-   Promiscuous terpene synthases from Prunella vulgaris highlight the    importance of substrate and compartment switching in terpene    synthase evolution. New Phytologist: nph.15778.-   Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: Molecular    Evolutionary Genetics Analysis across Computing Platforms (F U    Battistuzzi, Ed.). Molecular Biology and Evolution 35: 1547-1549.-   Kumar S, Stecher G, Tamura K. 2016. MEGA7: Molecular Evolutionary    Genetics Analysis Version 7.0 for Bigger Datasets. Molecular Biology    and Evolution 33: 1870-1874.-   Livak K J. Schmittgen T D. 2001. Analysis of Relative Gene    Expression Data Using Real-Time Quantitative PCR and the 2-ΔΔCT    Method. Methods 25: 402-408.-   Lopez-Gallego F, Agger S A, Abate-Pella D, Distefano M D,    Schmidt-Dannert C. 2010a. Sesquiterpene Synthases Cop4 and Cop6 from    Coprinus cinereus: Catalytic Promiscuity and Cyclization of Farnesyl    Pyrophosphate Geometric Isomers. ChemBioChem 11: 1093-1106.-   Lopez-Gallego F, Wawrzyn G, Schmidt-Dannert C. 2010b. Selectivity of    Fungal Sesquiterpene Synthases: Role of the Active Site's H-I Loop    in Catalysis. Applied and Environmental Microbiology 76: 7723-7733.-   McCormick S P, Alexander N J, Harris U. 2010. CLM1 of Fusarium    graminearum Encodes a Longiborneol Synthase Required for Culmorin    Production. Applied and Environmental Microbiology 76: 136-141.-   Medjahed F, Merouane A, Saadi A, Bader A, Cioni P L,    Flamini G. 2016. Chemical profile and antifungal potential of    essential oils from leaves and flowers of Salvia algeriensis    (Desf.): A comparative study. Chilean journal of agricultural    research 76: 195-200.-   Menotta M. Gioacchini A M, Amicucci A, Buffalini M, Sisti D.    Stocchi V. 2004. Headspace solid-phase microextraction with gas    chromatography and mass spectrometry in the investigation of    volatile organic compounds in an ectomycorrhizae synthesis system.    Rapid Communications in Mass Spectrometry 18: 206-210.-   Meshram V, Saxena S, Kapoor N. 2014. <I>Muscodor strobelii</I>, a    new endophytic species from South India. Mycotaxon 128: 93-104.-   Minerdi D, Bossi S, Gullino M L, Garibaldi A. 2009. Volatile organic    compounds: A potential direct long-distance mechanism for    antagonistic action of Fusarium oxysporum strain MSA 35.    Environmental Microbiology 11: 844-854.-   Molitor A, Zajic D, Voll L M, Pons-Kuhnemann J, Samans B, Kogel K-H,    Waller F. 2011. Barley Leaf Transcriptome and Metabolite Analysis    Reveals New Aspects of Compatibility and Piriformospora    indica-Mediated Systemic Induced Resistance to Powdery Mildew. /1427    MPMI24: 1427-1439.-   Morrone D, Lowry L, Determan M K, Hershey D M, Xu M, Peters    R J. 2010. Increasing diterpene yield with a modular metabolic    engineering system in E. coli: Comparison of MEV and MEP isoprenoid    precursor pathway engineering. Applied Microbiology and    Biotechnology 85: 1893-1906.-   Padovan A, Keszei A, Kollner T G, Degenhardt J, Foley W J. 2010. The    molecular basis of host plant selection in Melaleuca quinquenervia    by a successful biological control agent. Phytochemistry 71:    1237-1244.-   Pinedo C. Wang C-M, Pradier J-M, Dalmais B, Choquer M, Le Pecheur P.    Morgant G, Collado I G, Cane D E, Viaud M. 2008. Sesquiterpene    Synthase from the Botrydial Biosynthetic Gene Cluster of the    Phytopathogen Botrytis cinerea. ACS Chemical Biology 3: 791-801.-   Quin M B, Michel S N, Schmidt-Dannert C. 2015. Moonlighting Metals:    Insights into Regulation of Cyclization Pathways in Fungal    A6-Protoilludene Sesquiterpene Synthases. ChemBioChem 16: 2191-2199.-   Ramirez J, Gilardoni G, Ram6n E. Tosi S, Picco A. Bicchi C,    Vidari G. 2018. Phytochemical Study of the Ecuadorian Species    Lepechinia mutica (Benth.) Epling and High Antifungal Activity of    Carnosol against Pyricularia oryzae. Pharmaceuticals 11: 33.-   Ray P, Chi M-H, Guo Y. Chen C, Adam C. Kuo A, LaButti K. Lipzen A,    Barry K W, Grigoriev I V., et al. 2018. Genome Sequence of the Plant    Growth Promoting Fungus Serendipita vermifera subsp. bescii: The    First Native Strain from North America. Phytobiomes Journal 2:    62-63.-   Scher J M, Speakman J-B, Zapp J, Becker H. 2004. Bioactivity guided    isolation of antifungal compounds from the liverwort Bazzania    trilobata (L.) S. F. Gray. Phytochemistry 65: 2583-2588.-   Schmidt-Dannert C. 2014. Biosynthesis of Terpenoid Natural Products    in Fungi. In: Advances in Biochemical Engineering/Biotechnology.    19-61.-   Schmittgen T D, Livak K J. 2008. Analyzing real-time PCR data by the    comparative C T method. Nature Protocols 3: 1101-1108.-   Schüffler A. 2018. Secondary Metabolites of Basidiomycetes. In:    Physiology and Genetics. Chain: Springer International Publishing,    231-275.-   Shishova E Y, Di Costanzo L, Cane D E, Christianson D W. 2007. X-ray    Crystal Structure of Aristolochene Synthase from Aspergillus terreus    and Evolution of Templates for the Cyclization of Farnesyl    Diphosphate †, ‡. Biochemistry 46: 1941-1951.-   Trevizan L N F, Nascimento K F do, Santos J A, Kassuya C A L,    Cardoso C A L, Vieira M do C, Moreira F M F, Croda J, Formagio A    S N. 2016. Anti-inflammatory, antioxidant and anti-Mycobacterium    tuberculosis activity of viridiflorol: The major constituent of    Allophylus edulis (A. St.-Hil., A. Juss. &amp; Cambess.) Radlk.    Journal of Ethnopharmacology 192: 510-515.-   Varma A, Verma S, Sudha †, Sahay N, Bu{umlaut over ( )}    tehornbu{umlaut over ( )} bu{umlaut over ( )} tehorn B, Franken P.    1999. Piriformospora indica, a Cultivable Plant-Growth-Promoting    Root Endophyte Downloaded from.-   Wang X Y, Joshi Y, Hur J. 2011. The genus. 117: 405-422.-   Wawrzyn G T. Bloch S E, Schmidt-Dannert C. 2012a. Discovery and    Characterization of Terpenoid Biosynthetic Pathways of Fungi. In:    Methods in Enzymology. Elsevier Inc., 83-105.-   Wawrzyn G T, Quin M B, Choudhary S, López-Gallego F,    Schmidt-Dannert C. 2012b. Draft genome of omphalotus olearius    provides a predictive framework for sesquiterpenoid natural product    biosynthesis in basidiomycota. Chemistry and Biology 19: 772-783.-   Weiss M, Selosse M A, Rexer K H, Urban A, Oberwinkler F. 2004.    Sebacinales: A hitherto overlooked cosm of heterobasidiomycetes with    a broad mycorrhizal potential. Mycological Research.-   Wenke K, Kai M, Piechulla B. 2010. Belowground volatiles facilitate    interactions between plant roots and soil organisms. Planta 231:    499-506.-   Wu W. Tran W. Taatjes C A. Alonso-Gutierrez J, Lee T S, Gladden    J M. 2016. Rapid Discovery and Functional Characterization of    Terpene Synthases from Four Endophytic Xylariaceae (B Hamberger,    Ed.). PLOS ONE 11: e0146983.-   Xu L, Wang A, Wang J, Wei Q, Zhang W. 2017. Piriformospora indica    confers drought tolerance on Zea mays L. through enhancement of    antioxidant activity and expression of drought-related genes. The    Crop Journal 5: 251-258.-   Yuan X. 2005. Lichen-Like Symbiosis 600 Million Years Ago. Science    308: 1017-1020.-   Zuccaro A, Lahrmann U, Guldener U, Langen G, Pfiffi S, Biedenkopf D,    Wong P, Samans B. Grimm C, Basiewicz M, et al. 2011. Endophytic Life    Strategies Decoded by Genome and Transcriptome Analyses of the    Mutualistic Root Symbiont Piriformospora indica (BJ Howlett, Ed.).    PLoS Pathogens 7: e1002290.

All patents and publications referenced or mentioned herein areindicative of the levels of skill of those skilled in the art to whichthe invention pertains, and each such referenced patent or publicationis hereby specifically incorporated by reference to the same extent asif it had been incorporated by reference in its entirety individually orset forth herein in its entirety. Applicants reserve the right tophysically incorporate into this specification any and all materials andinformation from any such cited patents or publications.

The following statements are intended to describe and summarize variousfeatures of the invention according to the foregoing descriptionprovided in the specification and figures.

Statements:

-   -   1. An expression system comprising at least one expression        cassette having a heterologous promoter operably linked to a        nucleic acid segment encoding an enzyme that catalyzes synthesis        of viridiflorol.    -   2. An expression system comprising at least one expression        cassette having a heterologous promoter operably linked to a        nucleic acid segment encoding an enzyme with at least 90%        sequence identity to SEQ ID NO:1, 3, 4, or 5.    -   3. The expression system of statement 1 or 2, wherein at least        one expression cassette is within at least one expression        vector.    -   4. The expression system of statement 1, 2 or 3, wherein the        expression system further comprises one or more expression        cassettes having a promoter operably linked to a nucleic acid        segment encoding an enzyme that can synthesize isopentenyl        diphosphate (IPP), dimethylallyl diphosphate (DMAPP), or        geranylgeranyl diphosphate (GGPP).    -   5. The expression system of statement 1-3 or 4, wherein the        expression system has at least one expression cassette having a        constitutive promoter.    -   6. The expression system of statement 1-3 or 4, wherein the        expression system has at least one expression cassette having an        inducible promoter.    -   7. The expression system of statement 1-5 or 6, wherein the        expression system has at least one expression cassette having        the heterologous promoter is a lac promoter, a T7 promoter, a        Serendipita indica FCGB1 promoter, CaMV 35S promoter, CaMV 19S        promoter, nos promoter, Adh1 promoter, sucrose synthase        promoter, α-tubulin promoter, ubiquitin promoter, actin        promoter, cab promoter, PEPCase promoter, R gene complex        promoter, CYP71D16 trichome-specific promoter, CBTS        (cembratrienol synthase) promotor, Z10 promoter from a 10 kD        zein protein gene, Z27 promoter from a 27 kD zein protein gene,        plastid rRNA-operon (rrn) promoter, light inducible pea rbcS        gene, RUBISCO-SSU light-inducible promoter (SSU) from tobacco,        or rice actin promoter.    -   8. A host cell comprising the expression system of statement 1-6        or 7, which expression system is heterologous to the host cell.    -   9. The host cell of statement 8, which is a plant cell, an algae        cell, a fungal cell, a bacterial cell, or an insect cell.    -   10. The host cell of statement 8 or 9, which is a fungal cell or        a bacterial cell.    -   11. A method of synthesizing a terpene comprising incubating a        host cell that has the expression system of any of statements        1-7.    -   12. A method for synthesizing a terpene comprising incubating a        host cell comprising a heterologous expression system that        includes at least one expression cassette having a heterologous        promoter operably linked to a nucleic acid segment encoding an        enzyme with at least 90% sequence identity to SEQ ID NO:1, 3, 4,        or 5.    -   13. A method for synthesizing a terpene comprising incubating a        terpene precursor with an enzyme with at least 90% sequence        identity to SEQ ID NO: 1,3,4, or 5.    -   14. The method of statement 11, 12, or 13, further comprising        isolating one or more terpenes.    -   15. The method of statement 11-13 or 14, wherein the terpene        synthesized and/or isolated is viridiflorol.

-   -   16. A reaction mixture comprising one or more of the following:

The specific methods, expression systems, and compositions describedherein are representative of preferred embodiments and are exemplary andnot intended as limitations on the scope of the invention. Otherobjects, aspects, and embodiments will occur to those skilled in the artupon consideration of this specification and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, or limitation or limitations,which is not specifically disclosed herein as essential. The methods andprocesses illustratively described herein suitably may be practiced indiffering orders of steps, and the methods and processes are notnecessarily restricted to the orders of steps indicated herein or in theclaims.

Under no circumstances may the patent be interpreted to be limited tothe specific examples or embodiments or methods specifically disclosedherein. Under no circumstances may the patent be interpreted to belimited by any statement made by any Examiner or any other official oremployee of the Patent and Trademark Office unless such statement isspecifically and without qualification or reservation expressly adoptedin a responsive writing by Applicants.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any equivalent of the featuresshown and described or portions thereof, but it is recognized thatvarious modifications are possible within the scope of the invention asclaimed. Thus, it will be understood that although the present inventionhas been specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims and statements of theinvention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

1. An expression system comprising at least one expression cassettehaving a heterologous promoter operably linked to a nucleic acid segmentencoding an enzyme with at least 95% sequence identity to SEQ ID NO:1,3, 4, or
 5. 2. The expression system of claim 1, wherein theheterologous promoter is a bacterial, plant, or fungal promoter.
 3. Theexpression system of claim 1, wherein the heterologous promoter is a lacpromoter, a T7 promoter, a Serendipita indica FCGB1 promoter, CaMV 35Spromoter, CaMV 19S promoter, nos promoter, Adh1 promoter, sucrosesynthase promoter, α-tubulin promoter, ubiquitin promoter, actinpromoter, cab promoter, PEPCase promoter, R gene complex promoter,CYP71D16 trichome-specific promoter, CBTS (cembratrienol synthase)promotor, Z10 promoter from a 10 kD zein protein gene, Z27 promoter froma 27 kD zein protein gene, plastid rRNA-operon (rrn) promoter, lightinducible pea rbcS gene, RUBISCO-SSU light-inducible promoter (SSU) fromtobacco, or rice actin promoter.
 4. A host cell comprising theexpression system of claim 1, which expression system is heterologous tothe host cell.
 5. The host cell of claim 4, which is a plant cell, analgae cell, a fungal cell, a bacterial cell, or an insect cell.
 6. Amethod for synthesizing a terpene comprising incubating a host cell or aplant, wherein the host cell or the plant comprises a heterologousexpression system that includes at least one expression cassette havinga heterologous promoter operably linked to a nucleic acid segmentencoding an enzyme with at least 90% sequence identity to SEQ ID NO:1,3, 4, or
 5. 7. A method for synthesizing a terpene comprising incubatinga reaction mixture comprising a terpene precursor with an enzyme havingat least 90% sequence identity to SEQ ID NO: 1, 3, 4, or
 5. 8. Themethod of claim 6, further comprising isolating viridiflorol from thehost cell, the plant, or the reaction mixture.
 9. The method of claim 7,further comprising isolating viridiflorol from the host cell, the plant,or the reaction mixture.